2019010820198681112FFB7FFF7-3E69-FFFB-FFD8-FF95FFBEB2413364168ED7FD98F-964D-402F-AB70-5FC9B4CA48512305201815042019Nadia B. Páez, Santiago R. RonThis is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Abstract

Pristimantis is the most diverse genus of tetrapods comprising 532 described species. It contains a large number of morphologically cryptic species that are being discovered with the assistance of genetic evidence. We use molecular, morphological, bioacoustic, and environmental data to assess the phylogenetic relationships and determine the species within an Andean clade of Pristimantis, which is distributed from central Ecuador to northern Peru. We assign to this clade the name Huicundomantis and propose it as a subgenus. Our results show that Huicundomantis is composed of two large clades which we name as the P.phoxocephalus species group and the P.cryptomelas species group. Huicundomantis is composed of 28 species of which 12 have been described and 16 are new. We describe 11 of these undescribed species. The most effective characters to discriminate among species are DNA sequences, qualitative morphology, and advertisement calls. Morphometric and environmental characters are not very useful to define species limits. We clarify the identity of P.riveti and show that populations from southern Ecuador traditionally ascribed to P.riveti are a new species, P.lutzaesp. nov. We also show that P.prometeii is a junior synonym of P.hampatusami. The current diversity and geographic distribution of Huicundomantis are consistent with a model of allopatric speciation. All species have a restricted distribution range (less than 4330 km2) and are assigned to the Red List categories Data Deficient or threatened with extinction. We provide new reasons to increase conservation efforts for these species and their habitat. Taking our results into account, Pristimantis species richness in Ecuador increases from 211 to 221 species, and the number of species endemic to Ecuador from 119 to 129.

Pristimantis Jiménez de la Espada, 1870 is the most speciose genus of tetrapods with 532 described species (Frost 2019). It is distributed from Honduras in Central America to northern Argentina and southern Brazil, from the sea level to elevations above 4000 m a.s.l. (Frost 2019). In Ecuador, it comprises 34.3% of the anuran diversity with 204 species, of which 114 are endemic to the country (Ron et al. 2019).

Pristimantis taxonomy has been complex and labile because early studies were based almost exclusively on external morphology (e.g., Lynch and Duellman 1997; Lynch and Duellman 1980; Duellman and Lehr 2009), which was frequently insufficient to infer species limits and phylogenetic relationships. Recent studies have shown that species richness of Pristimantis is underestimated due to the existence of morphologically cryptic species (e.g., Elmer and Cannatella 2009; Padial and de la Riva 2009; Hutter and Guayasamin 2015; Ortega et al. 2015). The use of DNA sequences in combination with morphological and behavioral data has allowed objective species delimitation in a framework known as ‘integrative taxonomy’ (Will et al. 2005; Bickford et al. 2007; Padial et al. 2010). This approach has been increasingly applied to Neotropical amphibians resulting in the discovery of large numbers of cryptic species (e.g., Ron et al. 2006; Fouquet et al. 2007; Elmer and Cannatella 2009; Vieites et al. 2009; Funk et al. 2012; Caminer and Ron 2014; Ortega et al. 2015).

Unfortunately, relatively few groups of Pristimantis have been subject of systematic reviews based on genetic characters. Among the species that await such review are Pristimantisphoxocephalus (Lynch, 1979) and its close relatives Pristimantisriveti (Despax, 1911), P.spinosus (Lynch, 1979), and Pristimantisversicolor (Lynch, 1979). Pristimantisphoxocephalus was described from the Pacific versant of the Andes of central and southern Ecuador; its type locality is Pilaló in Cotopaxi Province (central Ecuador) at an elevation of 2340 m (Lynch 1979). Duellman and Wild (1993) provided two records from the Huancabamba Cordillera in northern Peru. Lynch and Duellman (1997) published a species account and hypothesized a close relationship with P.eugeniae, P.nyctophylax, and P.subsigillatus. They also suggested the inclusion of these species in the P.lacrimosus species group. Duellman and Pramuk (1999) commented on the existence of morphological differences between Ecuadorian and Peruvian populations. Duellman and Lehr (2009) provided a species account, noted additional morphological differences between Ecuadorian and Peruvian populations, and recommended the use of molecular characters to test the conspecificity of Ecuadorian and Peruvian populations. As currently defined, P.phoxocephalus has a wide distribution range, occurring in the Andean slopes from central Ecuador to northern Peru between 1800 and 3400 m a.s.l. (Duellman and Lehr 2009; Ron et al. 2019).

Pristimantisriveti was described from a single specimen collected in Ecuador, El Mirador (Despax 1911). This species name was not associated with any population until Lynch (1969) incorrectly identified populations of P.curtipes as “P.riveti” (Lynch 1979). Subsequently, Lynch (1979) provided a species account including a detailed description of morphological variation, comparisons with similar species, and natural history. The species account was based on specimens collected near Cuenca, Azuay Province, in southern Ecuador. Remarkably, Lynch characterized the distribution range of P.riveti as “both Andean Cordilleras and the connecting nudos at elevations of 2620–3420 m surrounding the Cuenca hoya in southern Ecuador”, a range that excludes the type locality. Subsequent references to the species (e.g., Almendáriz and Orcés 2004; Székely et al. 2016) relied on the morphological characterization provided by Lynch (1979) and ascribed the binomen to populations from Azuay Province and the adjacent Cañar and Loja Provinces. Heinicke et al. (2007) included a sample of “P.riveti” in their phylogeny. The sample was also from Azuay Province. Lynch and Duellman (1980) assigned P.riveti to the P.unistrigatus assemblage, and Hedges et al. (2008) later assigned it to the P.unistrigatus species group. Padial et al. (2014) left it unassigned.

Pristimantisspinosus was described by Lynch (1979) with the type locality “Sapote, Provincia Morona-Santiago, Ecuador, 2470 m”. Lynch (1979) hypothesized a close relationship with P.nigrogriseus and P.cryptomelas. Heinicke et al. (2007) included in their phylogeny a sample from Ecuador, Morona-Santiago, 10.6 km west of Plan de Milagro. Hedges et al. (2008) assigned it to the Pristimantisunistrigatus species group, and Padial et al. (2014) left it unassigned.

Pristimantisversicolor was described by Lynch (1979) from specimens collected at “15 km E Loja, 2800 m” in Loja Province, southern Ecuador. Duellman and Pramuk (1999) provided a species account and reported populations from Cordillera del Cóndor in northern Peru. Heinicke et al. (2007) included a sample of P.versicolor in their phylogeny which showed a close relationship with P.riveti and P.phoxocephalus. Lynch (1979) hypothesized a close relationship with P.cajamarcensis and P.unistrigatus. Lynch and Duellman (1980) assigned it to the P.unistrigatus assemblage, and Hedges et al. (2008) later assigned it to the P.unistrigatus species group. As with P.riveti, Padial et al. (2014) left it unassigned.

Our study aims to determine the content, identity, and phylogenetic relationships of species within P.phoxocephalus and closely related species. Our systematic review includes the description of 11 new species of which 10 belong to the P.phoxocephalus species group and one to the P.cryptomelas species group. Our taxonomic review is based on genetic, morphologic, bioacoustic, and environmental information.

Materials and methodsSpecies sampling

We focused our analysis in P.phoxocephalus and closely related species (Pyron and Wiens 2011; Padial et al. 2014). We expanded our study group to include morphologically similar species and species shown to be closely related to P.phoxocephalus based on an unpublished phylogeny of Pristimantis obtained by SRR as part of a large-scale review of Ecuadorian Pristimantis.

Phylogenetic analyses

For the molecular phylogenetic analyses, we used sequences of 85 individuals ascribed to the species above and added sequences of 48 individuals as the outgroup (46 Pristimantis spp., Craugastortalamancae, Epipedobatesboulengeri). Our final dataset consists of sequences of 133 individuals from 68 localities. Sequences of 117 of these individuals were newly generated and obtained from tissues deposited at the genome bank of the Zoology Museum, Pontificia Universidad Católica del Ecuador (QCAZ), representing 25 described and at least 22 undescribed species of Pristimantis. The remaining sequences, representing 16 species, were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/genbank). Vouchers and GenBank accession numbers are shown in Table 1.

Table 1.

Genbank accession numbers for DNA sequences used in the phylogenetic analyses.

Species

Voucher

16S

ND1

RAG1

GenSeq Nomenclature

Craugastortalamancae

QCAZ 30675

MK881414

MK881414

MK881322

genseq-3

Epipedobatesboulengeri

QCAZ 42251

MK881437

NA

MK881337

genseq-4

P.appendiculatus

QCAZ 16365

MK881401

NA

MK881315

genseq-4

P.atillo sp. nov.

QCAZ 31946

MK881419

NA

MK881324

genseq-2

QCAZ 40584

MK881435

NA

MK881335

genseq-2

QCAZ 42485

MK881438

NA

MK881338

genseq-2

QCAZ 42486

MK881439

NA

MK881339

genseq-2

QCAZ 42488

MK881440

NA

MK881340

genseq-2

QCAZ 42489

MK881441

NA

MK881341

genseq-2

QCAZ 42492

MK881442

NA

MK881342

genseq-2

QCAZ 42496

MK881443

NA

MK881343

genseq-2

QCAZ 42498

MK881444

NA

MK881344

genseq-2

QCAZ 42499

MK881445

NA

MK881345

genseq-2

QCAZ 42500

MK881446

NA

MK881346

genseq-1

QCAZ 42501

MK881447

NA

MK881347

genseq-2

QCAZ 42503

MK881448

NA

MK881348

genseq-2

QCAZ 42505

MK881449

NA

MK881349

genseq-2

QCAZ 42506

MK881450

NA

MK881350

genseq-2

QCAZ 42512

MK881451

NA

MK881351

genseq-2

QCAZ 42543

MK881452

NA

MK881352

genseq-2

QCAZ 42548

MK881453

NA

MK881353

genseq-2

P.atratus

QCAZ 45580

MK881471

MK881471

MK881364

genseq-4

QCAZ 45645

MK881473

MK881473

MK881366

genseq-4

P.bicantus

QCAZ 31860

MK881417

NA

NA

genseq-4

QCAZ 31986

MK881420

NA

MK881325

genseq-4

P.cajamarcensis

KU 217845

EF493663

NA

NA

genseq-4

QCAZ 17142

MK881403

MK881403

MK881317

genseq-4

QCAZ 24648

MK881405

NA

NA

genseq-4

QCAZ 31474

MK881416

NA

NA

genseq-4

P.ceuthospilus

CORBIDI 4306

MK881397

MK881397

MK881311

genseq-4

P.chomskyi sp. nov.

QCAZ 45666

MK881476

MK881476

MK881369

genseq-2

QCAZ 45669

MK881477

MK881477

MK881370

genseq-1

P.cryptomelas

QCAZ 45612

MK881472

MK881472

MK881365

genseq-4

QCAZ 45660

MK881475

MK881475

MK881368

genseq-4

P.curtipes

QCAZ 17505

MK881404

MK881404

MK881318

genseq-4

QCAZ 39568

MK881429

MK881429

NA

genseq-4

QCAZ 39569

MK881430

MK881430

NA

genseq-4

P.eremitus

QCAZ 43392

MK881460

NA

NA

genseq-4

P.gagliardoi

QCAZ 42575

MK881456

NA

MK881355

genseq-4

QCAZ 46738

MK881480

NA

MK881372

genseq-3

P.glandulosus

QCAZ 40153

MK881431

MK881431

MK881333

genseq-4

P.gloria sp. nov.

KU 218035

EF493348

NA

NA

genseq-4

QCAZ 16448

MK881402

MK881402

MK881316

genseq-2

QCAZ 31455

MK881415

NA

NA

genseq-2

QCAZ 56463

MK881502

NA

NA

genseq-2

QCAZ 57201

MK881503

NA

NA

genseq-1

P.hampatusami

QCAZ 58042

MK881504

MK881504

MK881387

genseq-3

P.jimenezi sp. nov.

QCAZ 45170

MK881466

MK881466

MK881360

genseq-1

QCAZ 45178

MK881468

MK881468

MK881362

genseq-2

QCAZ 46977

MK881481

MK881481

MK881373

genseq-2

QCAZ 46978

MK881482

MK881482

MK881374

genseq-2

P.kichwarum

QCAZ 25766

JN991458

NA

JQ025196

genseq-4

P.latidiscus

QCAZ 17101

EF493354

NA

EF493440

genseq-4

QCAZ 25852

JN991451

NA

JQ025188

genseq-4

P.lividus

QCAZ 54322

MK881497

MK881497

MK881382

genseq-4

P.lutzae sp. nov.

QCAZ 27471

MK881410

NA

NA

genseq-2

QCAZ 27534

MK881411

NA

NA

genseq-2

QCAZ 27597

MK881412

NA

NA

genseq-2

QCAZ 32785

MK881421

MK881421

MK881326

genseq-2

QCAZ 32786

MK881422

MK881422

MK881327

genseq-2

QCAZ 32791

MK881424

MK881424

MK881329

genseq-2

QCAZ 37546

MK881428

NA

NA

genseq-2

QCAZ 47211

MK881487

NA

NA

genseq-2

QCAZ 53728

MK881495

NA

NA

genseq-1

P.multicolor sp. nov.

QCAZ 47213

MK881488

NA

NA

genseq-1

QCAZ 47214

MK881489

NA

NA

genseq-2

P.muscosus

QCAZ 54857

MK881501

MK881501

MK881386

genseq-4

P.nangaritza sp. nov.

QCAZ 41710

MK881436

MK881436

MK881336

genseq-1

P.omeviridis

QCAZ 10564

MK881398

NA

MK881312

genseq-4

P.petrobardus

KU 212293

EF493367

NA

NA

genseq-2

P.philipi

KU 217863

EF493672

NA

NA

genseq-3

QCAZ 37528

MK881425

MK881425

MK881330

genseq-3

QCAZ 37537

MK881426

MK881426

MK881331

genseq-3

QCAZ 37542

MK881427

MK881427

MK881332

genseq-3

P.phoxocephalus

QCAZ 58463

MK881507

MK881507

MK881390

genseq-3

P.pichincha

QCAZ 11673

MK881399

MK881399

MK881313

genseq-4

P.pycnodermis

KU 218028

EF493680

NA

NA

genseq-4

KU 218030

EF493683

NA

NA

genseq-4

P.quaquaversus

QCAZ 25676

JN991463

NA

JQ025201

genseq-4

P.rubicundus

QCAZ 26551

MK881407

MK881407

MK881320

genseq-4

P.sobetes

QCAZ 11737

MK881400

NA

MK881314

genseq-4

P.spinosus

KU 218052

EF493673

NA

NA

genseq-4

P.sternothylax

CORBIDI 13316

MK881393

MK881393

MK881308

genseq-4

P.teslai sp. nov.

QCAZ 46213

MK881478

NA

NA

genseq-1

P.thymelensis

QCAZ 16428

EF493516

NA

EF493442

genseq-4

QCAZ 62801

MK881508

MK881508

MK881391

genseq-4

QCAZ 62803

MK881509

MK881509

MK881392

genseq-4

P.tinguichaca

QCAZ 31945

MK881418

MK881418

MK881323

genseq-3

QCAZ 40581

MK881432

NA

NA

genseq-3

QCAZ 40582

MK881433

NA

MK881334

genseq-3

QCAZ 42570

MK881454

NA

NA

genseq-3

QCAZ 42574

MK881455

NA

MK881354

genseq-3

QCAZ 42576

MK881457

NA

MK881356

genseq-3

QCAZ 42578

MK881458

NA

MK881357

genseq-3

QCAZ 54601

MK881498

NA

MK881383

genseq-3

QCAZ 54602

MK881499

NA

MK881384

genseq-3

QCAZ 54603

MK881500

NA

MK881385

genseq-3

P.torresi sp. nov.

QCAZ 47342

MK881490

NA

NA

genseq-1

QCAZ 47397

MK881492

MK881492

MK881380

genseq-2

P.totoroi sp. nov.

KU 218025

EF493349

NA

NA

genseq-4

QCAZ 25105

MK881406

MK881406

MK881319

genseq-1

QCAZ 58425

MK881505

MK881505

MK881388

genseq-2

P.unistrigatus

KU 218057

EF493387

NA

EF493444

genseq-4

P.verrucolatus sp. nov.

QCAZ 45174

MK881467

MK881467

MK881361

genseq-2

QCAZ 45195

MK881469

MK881469

MK881363

genseq-2

QCAZ 46982

MK881483

MK881483

MK881375

genseq-1

QCAZ 46992

MK881484

MK881484

MK881376

genseq-2

QCAZ 46993

MK881485

MK881485

MK881377

genseq-2

P.versicolor

KU 218096

EF493389

NA

EF493431

genseq-4

QCAZ 45650

MK881474

MK881474

MK881367

genseq-4

QCAZ 46310

MK881479

MK881479

MK881371

genseq-4

P.wiensi

KU 219796

EF493668

NA

NA

genseq-2

P.yumbo

QCAZ 52241

MK881494

NA

MK881381

genseq-3

QCAZ 58450

MK881506

MK881506

MK881389

genseq-4

Pristimantis sp. (CCS1)

QCAZ 32790

MK881423

MK881423

MK881328

genseq-4

Pristimantis sp. (CCS2)

QCAZ 45129

MK881462

MK881462

MK881358

genseq-4

QCAZ 45155

MK881465

MK881465

MK881359

genseq-4

Pristimantis sp. (UCS1)

QCAZ 53999

MK881496

NA

NA

genseq-4

Pristimantis sp. (UCS2)

QCAZ 45029

MK881461

NA

NA

genseq-4

Pristimantis sp. (UCS3)

QCAZ 26642

MK881409

NA

NA

genseq-4

Pristimantis sp.

CORBIDI 14804

MK881394

MK881394

MK881309

genseq-4

CORBIDI 14805

MK881395

NA

NA

genseq-4

CORBIDI 2848

MK881396

MK881396

MK881310

genseq-4

QCAZ 26586

MK881408

NA

MK881321

genseq-4

QCAZ 29206

MK881413

NA

NA

genseq-4

QCAZ 40583

MK881434

NA

NA

genseq-4

QCAZ 43024

MK881459

NA

NA

genseq-4

QCAZ 45131

MK881463

NA

NA

genseq-4

QCAZ 45142

MK881464

NA

NA

genseq-4

QCAZ 45210

MK881470

NA

NA

genseq-4

QCAZ 47002

MK881486

MK881486

MK881378

genseq-4

QCAZ 47353

MK881491

MK881491

MK881379

genseq-4

QCAZ 50732

MK881493

NA

NA

genseq-4

DNA was extracted from liver or muscle tissue preserved in 95% ethanol or tissue storage buffer using a guanidine thiocyanate protocol (M. Fujita, unpublished) with some modifications. Primers used for the PCR amplification of the below-mentioned genes are listed in Suppl. material 1. Amplicons were sequenced by the Macrogen Sequencing Team (Macrogen Inc., Seoul, Korea).

The phylogenetic analyses were based on a 3199 bp dataset containing DNA sequences of the mitochondrial genes 16S (1318 bp, partial sequence), tRNALeu (90 bp), NADH dehydrogenase subunit 1 ND1 (961 bp), tRNAIle (73 bp), tRNAGln (71 bp), and tRNAMet (31 bp), and the nuclear gene RAG1 (655 bp). The alignment of the sequences was performed in GeneiousPro v. 5.4.6 (GeneMatters Corp.) with the plug-in MAFFT (Katoh et al. 2002) and a posterior manual alignment with Mesquite v. 3.02 (Maddison and Maddison 2014). The aligned matrix is available at http://zenodo.org under https://doi.org/10.5281/zenodo.2555411. The best partitioning scheme and the best-fit model of molecular evolution for each partition were estimated with the software PartitionFinder v. 1.1.1 (Lanfear et al. 2012) under the greedy algorithm and the Bayesian information criterion (BIC).

Phylogenetic analyses were performed under two approaches, Bayesian Inference (BI) and Maximum-likelihood (ML). The Bayesian analysis was carried out with MrBayes v. 3.2.1 (Ronquist et al. 2012) in two independent searches for 3×107 generations; each search had four Monte Carlo Markov Chains. The temperature parameter was set at 0.5. Trees were sampled every 1000 generations. We evaluated the convergence of the chains with software Tracer v. 1.6. (Rambaut et al. 2014) by inspecting plots of parameter values vs generation and confirming stationarity in their values. We also evaluated the Effective Sample Size and considered the search finished when all values were greater than 200. We discarded 25% of the 30000 trees as burn-in. The remaining trees were combined to obtain a 50% majority-rule consensus tree as well as the branch posterior probabilities (BPP).

For the ML analysis, we used Garli v. 2.0 (Zwickl 2006). We run ten replicate searches starting from stepwise trees and 10 from random trees. We finished the search after 100000 generations without topology improvements (genthreshfortopoterm = 100000); we used default values for the remaining parameters. Non-parametric bootstrap values (NPB) were obtained with 500 pseudoreplicates with the same settings of the random search, each with two independent searches. The 50% majority rule consensus for the bootstrap trees was obtained with Mesquite v. 2.75. We used the same settings to run two additional ML analyses, one exclusively for the nuclear gene and one for the mitochondrial genes. Throughout the text, we considered that a node has “strong support” when its bootstrap value is >70 and its Bayesian posterior probability is 0.95 or higher.

In order to identify candidate species, we calculated uncorrected p genetic distances for gene 16S (~1300 bp), within and between clades, with software MEGA v.7.0 (Kumar et al. 2016). See Integrative Analyses section for details.

Morphological analyses

Specimens deposited at the QCAZ collection were preserved in 10% formalin and stored in 70% ethanol. Photographs of preserved specimens were taken while these were submersed in ethanol to avoid reflections. We compared qualitative and quantitative morphological characters between clades. We used only adult specimens for these analyses but, when available, juveniles were considered to describe morphological variation. We examined 750 individuals from central and southern Ecuador ranging from 230 to 4200 m a.s.l. (Figs 1, 2), from collections of the Zoology Museum, Pontificia Universidad Católica del Ecuador (QCAZ), Ecuadorian Museum of Natural Sciences (MECN), Museum of Natural History, University of Kansas, USA (KU), Gustavo Orcés Museum of Natural History, Escuela Politécnica Nacional, Ecuador (MEPN), National Museum of Natural History, Smithsonian Institution, USA (USNM), and French Museum of Natural History of Paris (MNHNP); information for each individual is detailed in Suppl. material 2.

We determined sex and reproductive condition by looking for vocal sacs, vocal slits, and nuptial pads, and by gonadal inspection. For males, the presence of vocal sac, vocal slits, or nuptial pads was used as indicator of adulthood; when these characters were absent, we examined the gonads and categorized an individual as adult if the testes were swollen and enlarged. For females, we considered the presence of convoluted oviducts and large ovarian eggs as features of adulthood (Duellman and Lehr 2009).

Diagnosis and description of species follow Duellman and Lehr (2009) terminology. We use the term “distinct” for a character that is clearly visible, and “indistinct” for a character that is barely visible. When referring to the tympanum state, we use “prominent” to indicate that it protrudes from the skin surface; when referring to tubercles, we use “prominent” to imply they are conspicuous and elevated. Following Lynch and Duellman (1997), we qualify Toe V as slightly longer than Toe III when the distal end of Toe V does not reach the proximal edge of the distal subarticular tubercle on Toe IV when they are adpressed. Toe V is called longer than Toe III when its distal end reaches the proximal edge of the distal subarticular tubercle in Toe IV and does not extend to its distal edge. Toe V is much longer than Toe III if the distal end of Toe V reaches, or extends beyond, the distal edge of the distal subarticular tubercle on Toe IV. Abbreviations used for quantitative descriptive characters are: snout-vent length (SVL, distance from the tip snout to posterior margin of vent); head width (HW, greatest width of head); head length (HL, distance from the tip of snout to posterior angle of jaw articulation); eye diameter (ED, distance between anterior and posterior borders of eye); interorbital distance (IOD); upper eyelid width (EW); eye-nostril distance (EN); internarial distance (IND); tympanic diameter (TD, distance between external anterior and posterior margins of tympanic annulus); tympanum-eye distance (TED, distance between anterior margin of the tympanum to the closest point of the posterior margin of the eye); tibia length (TL, length of flexed leg from knee to heel); foot length (FL, distance from heel to tip of toe IV). All measurements were taken with a digital caliper with a precision of 0.01 mm.

For morphometric analyses, we measured 232 individuals of the candidate species and P.phoxocephalus and P.versicolor. We performed separate analyses for males and females. As they have been reported to be variable among Pristimantis species (Arroyo et al. 2005), we used SVL, HL, HW, TD, and ED measurements for morphometrics. To assess the degree of morphometric differentiation between candidate species, we performed a Discriminant Function Analysis (DFA) including the variables mentioned above without size correction. For pairwise comparisons, we used the Wilcoxon signed-rank test with corrected measurements. To remove the effect of co-varying body size, we made linear regressions between each variable and SVL. Statistical analyses were performed using software JMP v. 9.0.1 (SAS Institute Inc. 2010).

Bioacoustic analyses

We analyzed recordings of eight individuals belonging to four candidate species of the clade under study, namely Pristimantisjimenezi sp. nov., Pristimantisphoxocephalus, Pristimantistotoroi sp. nov, and Pristimantisverrucolatus sp. nov. (see Results).

Recordings were obtained from the QCAZ audio library and are available in BIOWEB (http://bioweb.bio/faunaweb/amphibiaweb/). Recording temperatures ranged between 8.8 and 12 °C. Recordings of P.phoxocephalus were obtained at its type locality, Pilaló, Cotopaxi Province. We recorded one collected and two non-collected individuals from the same chorus. Their calls had similar properties; however, we did not include the advertisement call of the collected individual in the analysis to avoid accounting for effects of stress on the characteristics of the call given that it was recorded from a plastic bag after the individual’s capture. Advertisement calls for P.jimenezi sp. nov. were recorded from Azuay Province at two localities: at the border of Cajas National Park (QCAZ 45178) and Zadracay River (QCAZ 46977) (see Suppl. material 2 for details on localities); both individuals were sequenced. For P.verrucolatus sp. nov., we had recordings available for two individuals (QCAZ 46984, QCAZ 46981) from Shoupshe, Azuay Province. Finally, recordings for P.totoroi sp. nov. were obtained from Bosque Protector Cashca Totoras, Bolívar Province. We recorded a non-collected and a collected individual (QCAZ 25105), which was subsequently sequenced.

We analyzed calls with the software Raven Pro v. 1.3 (Cornell Lab of Ornithology 2003–2008) using a sampling rate of 44.1 kHz and a frequency resolution of 10.8–11.2 Hz. Temporal data was measured on oscillograms and frequency data on power spectrums (e.g., Caminer and Ron 2014). We randomly chose five advertisement calls from each recording and measured: (1) number of notes per call; (2) number of harmonics; (3) note duration; (4) inter-note interval; (5) peak time of the note (time from beginning of the note to peak energy); (6) dominant frequency; (7) frequency of the second harmonic; (8) initial and (9) final frequency of the note; and (10) frequency change in each note, estimated from the difference in frequency between the beginning and end of a note. Because of the low number of individuals recorded per species, we could not apply statistical comparisons. However, each species had a highly stereotyped call, qualitatively distinct from the others.

Environmental analyses

We considered environmental differentiation as additional evidence to define species boundaries (e.g., Rissler and Apodaca 2007; Ortega et al. 2015). Accordingly, we analyzed differences in bioclimatic and vegetation-related variables between candidate species.

To avoid spatial autocorrelation, we reduced the number of localities using a buffer with a 5 km radius around each point; if two or more localities of the same species were at a distance <10 km from each other, we randomly chose one of them. We obtained a total of 62 localities for the analysis. As species limits in this clade are incorrectly defined, we did not include geographic records from the literature. Locality data are available in Suppl. material 2. A distribution map is presented in Figures 1, 2.

For the description of the habitat and distribution of the species, we assigned natural regions according to Ron et al. (2019); we estimated the Extent of Occurrence and Area of Occupancy of each species with the R package red (Cardoso 2017).

Integrative analyses

To determine species limits, we examined the covariation of independent sets of characters under the framework of integrative taxonomy (Dayrat 2005; Vieites et al. 2009). Our datasets consisted of genetic, morphological, bioacoustic, and environmental characters. We assumed that covariation between independent sets of characters is an indication of evolutionary independence between lineages (Vieites et al. 2009).

Based on the correspondence between morphological and genetic divergence, we set a 0.02 threshold of uncorrected p distances for the gene 16S to identify candidate species. We chose a lower threshold than that proposed by Fouquet et al. (2007) for amphibians (0.03) because there is evidence indicating that sister species in Pristimantis (e.g., Ortega et al. 2015; Padial and de la Riva 2009) and other Neotropical frogs (e.g., Caminer and Ron 2014; Jungfer et al. 2013; Coloma et al. 2012; Funk et al. 2012) often differ by genetic distances <0.03. Candidate species categories follow Vieites et al. (2009). Each candidate species was categorized as: (1) unconfirmed candidate species (UCS) when genetic distance was >0.02 and there were no additional character sets available; (2) confirmed candidate species (CCS) when genetic distance was >0.02 and there was covariation between genetic and morphological, bioacoustic and/or environmental characters; (3) deep conspecific linage (DCL) when genetic distance was >0.02 and there was not covariation between genetic and morphological, bioacoustics, and environmental characters. We followed the species concept proposed by de Queiroz (2007) that defines a species as a separately evolving metapopulation lineage.

ResultsPhylogeny and genetic variation

The best partition scheme for the concatenated matrix consisted of the following five partitions and models: 16S, ND1 1st position (GTR+I+G); ND1 2nd position (HKY+I+G); ND1 3rd position (TrN+I+G); RAG1 3rd position (K80+G); RAG1 1st and 2nd position (K81uf+I+G).

Tree topologies under ML and Bayesian inference were similar except for the order of divergence events of P.tinguichaca, P. sp. CCS1, and P.atillo sp. nov. (see Integrative results). The best ML tree based on nuclear and mitochondrial genes is shown in Figure 3; the consensus tree of the Bayesian analysis is available through zenodo.org (http://doi.org/10.5281/zenodo.2556418). We found a strongly supported clade (94 NPB, 1.0 BBP) that has P.philipi diverging basally and sister to a clade composed of two large subclades with strong support (Fig. 3 and Suppl. material 3). We are referring to these clades as the P.phoxocephalus and P.cryptomelas species groups. Figure 3 shows the species limits identified in the integrative analyses (see below). The topology of the ML tree based on mitochondrial genes was congruent with the one based on the complete dataset for branches with strong support (>75% NBP) (Suppl. material 4). The tree based on the nuclear gene (Suppl. material 5) has low resolution and low branch support.

Uncorrected p-genetic distances are summarized in Tables 2, 3. Average interspecific genetic distances (16S) for the ingroup range from 2.0 to 12.9% (1.8 to 5.9% between individuals of sister species). Pristimantisgloria sp. nov. was the species with the highest divergence between two populations; the population from Loja Province presented up to 1.3% of genetic distance with populations from Azuay Province (average intraspecific distance: 0.7%). We found 26 clades (i.e., candidate species) with genetic distances >2.0%. Ten of these clades are formerly described species: P.atratus (Lynch, 1979), P.cryptomelas (Lynch, 1979), P.gagliardoi Bustamante & Mendelson, 2008, P.prometeiiSzékely et al., 2016, P.muscosus (Duellman & Pramuk, 1999), P.philipi, P.phoxocephalus, P.spinosus, P.tinguichacaBrito et al., 2016, and P.versicolor; the remaining 16 clades are candidate species to be confirmed with the following analyses.

Table 2.

Genetic divergence (Gene 16S) between species of the Pristimantisphoxocephalus species group. Mean uncorrected p distances (%) between groups are shown under the diagonal, standard error estimates above the diagonal. Mean uncorrected p distance within each clade and its standard error are shown on the diagonal. Number of samples is given in brackets after the name of each clade. Standard error estimates were obtained by a bootstrap procedure in MEGA 7.0.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

1

P.atillo (18)

0

0.9

0.9

0.7

0.9

1.0

0.8

0.9

1.0

0.6

0.8

1.0

0.9

0.8

0.7

0.8

0.8

0.5

0.7

0.9

2

P.atratus (2)

7.3

0

0.7

0.8

1.0

0.7

0.8

0.8

0.8

1.0

1.0

0.9

0.7

0.6

0.8

0.7

0.7

0.9

1.0

1.0

3

P.chomskyi (2)

7.1

5.4

0.2±0.1

0.7

1.0

0.7

0.7

0.8

0.8

1.0

1.0

0.9

0.7

0.7

0.7

0.7

0.7

0.9

1.0

1.0

4

P.gloria (5)

5.1

5.8

6.3

0.7±0.2

0.9

0.7

0.6

0.8

0.8

0.8

0.9

0.9

0.7

0.7

0.7

0.7

0.4

0.8

0.9

0.9

5

P.hampatusami (1)

7.0

11.1

10.9

8.7

NA

1.0

0.9

1.0

1.0

1.0

0.9

1.0

1.0

0.9

0.9

1.0

0.9

1.0

1.1

0.7

6

P.jimenezi (4)

7.7

6.8

7.8

5.8

10.7

0.5±0.2

0.7

1.0

0.9

1.0

1.0

0.9

0.8

0.6

0.7

0.7

0.6

1.0

1.1

1.0

7

P.lutzae (9)

5.0

5.7

5.6

2.9

7.5

5.3

0.2±0.1

0.9

0.8

0.8

0.8

0.9

0.7

0.6

0.7

0.7

0.6

0.8

0.9

0.9

8

P.multicolor (2)

6.7

4.4

4.9

5.5

8.9

6.7

5.5

0

0.9

1.0

1.0

1.0

0.9

0.9

1.0

0.9

0.8

1.0

1.0

1.0

9

P.phoxocephalus (1)

7.8

5.8

6.7

5.4

11.1

6.8

5.8

6.3

NA

1.1

1.0

1.0

0.7

0.8

0.9

0.8

0.9

1.0

1.1

1.0

10

P.teslai (1)

2.6

7.6

7.4

5.9

7.6

7.9

5.2

7.1

8.0

NA

0.9

1.0

1.0

0.8

0.8

0.8

0.9

0.6

0.6

1.0

11

P.tinguichaca (10)

4.7

7.8

7.7

6.1

7.0

7.8

5.3

7.5

7.7

5.7

0

1.0

0.9

0.8

0.8

0.8

0.9

0.8

0.9

0.9

12

P.torresi (2)

8.7

8.7

9.5

7.3

11.1

9.1

7.5

8.3

8.6

9.1

8.6

0.9±0.4

0.9

0.9

0.8

0.9

0.8

1.0

1.1

1.1

13

P.totoroi (3)

7.0

6.0

6.8

6.0

11.1

6.9

5.5

6.4

5.9

7.4

7.2

8.0

1±0.2

0.7

0.7

0.8

0.7

0.9

0.9

0.9

14

P.verrucolatus (5)

4.6

5.4

6.6

4.8

9.7

5.6

4.3

6.0

6.1

4.9

4.9

8.6

5.7

0.2±0.1

0.6

0.5

0.6

0.8

0.8

0.9

15

P.versicolor (3)

3.9

7.2

7.4

6.0

10.2

7.5

5.6

7.9

8.1

4.4

5.8

8.7

7.1

4.7

0.38±0.1

0.6

0.6

0.7

0.8

1.0

16

Pristimantis CCS1 (1)

4.4

6.2

7.2

5.3

10.4

6.4

5.1

6.9

6.8

4.7

4.6

8.6

6.1

3.2

5.0

NA

0.6

0.8

0.9

0.9

17

Pristimantis CCS2 (2)

5.4

5.8

6.0

2.0

7.5

5.8

2.9

5.4

6.0

6.4

5.8

7.6

5.9

4.5

5.7

5.1

0.19±0.1

0.8

0.9

0.9

18

Pristimantis UCS1 (1)

2.5

7.0

7.2

5.4

7.4

7.5

5.1

6.5

7.3

3.0

5.6

8.6

7.4

4.3

2.9

4.8

5.8

NA

0.6

0.9

19

Pristimantis UCS2 (1)

3.2

7.7

7.3

6.3

8.0

8.4

5.9

6.9

8.1

2.5

5.8

9.9

7.4

4.9

4.3

4.8

6.6

2.8

NA

1.0

20

Pristimantis UCS3 (1)

6.9

8.1

7.4

6.6

3.5

7.3

6.2

8.0

7.7

7.1

6.5

8.5

7.7

6.9

7.5

6.7

6.8

7.2

7.7

NA

Table 3.

Genetic divergence (16S) between species of the Pristimantiscryptomelas species group. Mean uncorrected p distances (%) between groups are shown under the diagonal, standard error estimates above the diagonal. Mean uncorrected p distance within each clade and its standard error are shown on the diagonal. Number of samples is given in brackets after the name of each clade. Standard error estimates were obtained by a bootstrap procedure in MEGA 7.0.

1

2

3

4

5

1

P.cryptomelas (2)

0

1.1

0.8

0.9

0.6

2

P.gagliardoi (2)

8.8

0.9±0.4

1.1

1.1

1.0

3

P.muscosus (1)

7.6

8.6

NA

1.0

0.7

4

P.nangaritza (1)

8.1

8.3

9.8

NA

0.9

5

P.spinosus (1)

5.9

7.6

7.4

8.1

NA

Morphological analyses

Morphological variation is shown in Figure 4. The ingroup shares the absence of dorsolateral folds (except for P.atratus), presence of a tympanic annulus and membrane (except for P.philipi), dentigerous processes of vomers evident, first finger shorter than the second, presence of lateral fringes on fingers and toes, basal webbing on toes (except for P.philipi and P.prometeii), and small to prominent tubercles on heel. Additionally, all the species of the P.cryptomelas group bear postocular folds and prominent tubercles on eyelids and heels. Because postocular folds and prominent tubercles on eyelids and heels are absent in the P.phoxocephalus group and P.philipi, both characters appear to be synapomorphies for the P.cryptomelas group. Meanwhile, species of the Pristimantisphoxocephalus group have a keel or a papilla at the tip of the snout. The papilla is absent in P.philipi and all species of the P.cryptomelas group, except for P.cryptomelas which has papilla, suggesting that the presence of a papilla is a synapomorphy for the P.phoxocephalus group with an independent origin in P.cryptomelas. Traits that differ the most between species include snout shape, presence or absence of middorsal, lateral and postocular folds, dorsal skin texture, shape and width of discs, and coloration of groins, hidden surfaces of thighs, and iris. Character states for qualitative morphological traits are shown in Table 4.

Qualitative morphological traits of Pristimantis, subgenus Huicundomantis. Coloration corresponds to live individuals unless otherwise noticed. + is for present; – is for absent.

Dorsal texture

Cranial crests

Postocular/scapular fold/ridge

Middorsal fold

Tubercles row head

Dorsolateral folds

Lateral fold

Snout shape (dorsal; lateral)

Discs (expansion, shape)

Basal webbing

Iris coloration

Groin coloration

P.atillo sp. nov.

Shagreen with or without scattered small subconical tubercles

–

–

+/–

+

–

+/–

Acuminate; protruding; with a fleshy keel

Expanded to broadly expanded, rounded to elliptical

+

Copper with a faint medial horizontal darker streak

Bright orange, surrounded or not by yellow spots

P.atratus

Shagreen

Low

–

+/–

+/–

+

+

Subacuminate; rounded; with or without a papilla

Broadly expanded, rounded to elliptical

+

Pale yellow to pale bronze, thin brown streak

Black with white or yellow spots

P.balionotus

Tuberculate

–

–

–

–

–

+/–

Subacuminate; rounded; with a papilla

Slightly expanded, elliptical

–

Bronze with a red medial streak

In preservative, pale rusty brown with or without faint brown marbling

P.chomskyi sp. nov.

Shagreen

–

–

–

–

–

–

Subacuminate; rounded; with or without a papilla

Expanded, rounded to elliptical

+

Orange with a faint reddish brown streak

Dark chocolate brown with or without small cream flecks

P.gloria sp. nov.

Tuberculate to warty

–

–

+

–

–

–

Subacuminate; rounded; with or without a papilla

Expanded, truncate to elliptical

+

Light silver to cream with wide black reticulations and a red streak

Pinkish to purplish brown with irregular cream to light brown flecks or spots

P.hampatusami

Shagreen with scattered tubercles

–

Low, W-shaped

+

+

–

+

Subacuminate; rounded; with or without a papilla

Broadly expanded, elliptical to truncate

–

Golden to bronze with a red medial horizontal streak

Reddish brown with yellow spots

P.jimenezi sp. nov.

Shagreen with or without scattered small subconical tubercles

–

–

+

+

–

+

Acuminate; protruding; with a fleshy keel

Expanded to broadly expanded, elliptical to truncate

+

Copper with red medial streak to completely red

Pinkish, purplish or dark brown with small light brown to yellow spots

P.lutzae sp. nov.

Shagreen to tuberculate

–

–

+/–

+/–

–

+/–

Round to subacuminate, with or without a papilla

Expanded, elliptical to truncate

+

Golden to creamy brown with a reddish brown medial streak

Pinkish to reddish brown, suffused or not with orange, with cream or light brown spots

P.multicolor sp. nov.

Shagreen to warty

–

–

–

+/–

–

–

Subacuminate; rounded; with or without a papilla

Expanded to broadly expanded, elliptical to truncate

+

Copper to creamy yellow with or without a red to dark brown streak

Cream, orange, brown, or black with or without cream to yellow flecks or spots

P.phoxocephalus

Shagreen with scattered small tubercles

–

–

+

+

–

+/–

Acuminate; protruding; with a fleshy keel

Broadly expanded, rounded to elliptical

+

Golden with wide black reticulations

In preservative, brown with cream reticulation or spots

P.percultus

Tuberculate

Low

–

–

–

–

–

Subacuminate; acutely rounded; with a keel

Expanded, elliptical

+

Copper with or without a faint red medial streak

Yellow with black reticulations

P.teslai sp. nov.

Tuberculate with prominent and rounded tubercles

–

–

–

+

–

–

Acuminate; protruding; with a fleshy keel

Expanded to broadly expanded, elliptical to truncate

+

Copper

Dark brown with yellow irregular blotches

P.tinguichaca

Smooth

–

–

+

+

–

+

Subacuminate; rounded; with or without a papilla

Broadly expanded, rounded

+

Red with a dark medial streak

Reddish to dark brown

P.torresi sp. nov.

Shagreen

–

–

+

+/–

–

+

Acuminate; protruding; with a fleshy keel

Broadly expanded, elliptical to truncate

+

Golden to beige with red to reddish brown streak

Light purplish brown to brown with or without yellow spots

P.totoroi sp. nov.

Shagreen with or without scattered small tubercles

–

–

+

+

–

+

Acuminate; protruding; with a fleshy keel

Broadly expanded, rounded to elliptical

+

Golden with a medial horizontal red streak

In preservative, brown with or without pale spots

P.verrucolatus sp. nov.

Shagreen with or without scattered small tubercles

–

–

+/–

+/–

–

+

Acuminate; rounded; with a fleshy keel

Broadly expanded, elliptical to truncate

+

Coppery brown

Reddish brown with small light brown, orangey brown or yellow spots

P.versicolor

Shagreen to tuberculate

–

–

+/–

–

–

–

Subacuminate; rounded; with or without a papilla

Expanded, rounded to elliptical

+

Bronze-white with a reddish brown streak

Brown to black with cream to reddish pink flecks or spots

P.philipi

Tuberculate with low tubercles and warts

–

W-shaped ridge

+/–

+

–

–

Rounded

Slightly expanded, truncate

–

Grayish bronze

Black with or without white or yellow streaks

P.cryptomelas

Shagreen

–

Prominent,) (-shaped

+/–

+

–

+

Subacuminate; rounded; with or without a papilla

Broadly expanded, rounded to elliptical

+

Red, orange or cream with a red or orange streak

Black with or without yellow or white spots

P.gagliardoi

Shagreen with scattered tubercles

–

Prominent, W-shaped

+/–

+

–

+

Rounded

Broadly expanded, elliptical to truncate

+

Bronze with or without a brown medial horizontal streak

Pink to orange with or without brown blotches

P.muscosus

Smooth to shagreen

–

Low,) (-shaped

–

+

–

+

Rounded

Broadly expanded, elliptical to truncate

+

Reddish brown

Dark brown with orange, yellow or white spots

P.nangaritza sp. nov.

Finely tuberculate

–

Low,) (-shaped

+/–

+

–

+/–

Subacuminate; rounded

Broadly expanded, rounded to elliptical

+

Unknown

In preservative, light brown with or without pale flecks

P.spinosus

Finely tuberculate

Low

Low,) (-shaped

–

+

–

+

Subacuminate; rounded to truncate

Broadly expanded, rounded

+

Unknown

Black with big white spots

Ranges of quantitative variables widely overlap among species (Fig. 5A, B). However, several species pairs differ significantly from each other according to one or more variables (see Fig. 5A, C for an example); these results will be mentioned when useful to discriminate similar species. The DFA classification assigned the correct species in 77.1% of the specimens in females (64 out of 83), and 68.7% in males (126 out of 182). Although the DFA classification was not perfect, it shows some morphometric differentiation among species. Summarized information of morphometric measurements is shown in Table 5.

Morphological and environmental comparisons among species of Pristimantis, sugenus Huicundomantis. Intra and interspecific variation in tympanum size, respective to body, in A females and B males. Green horizontal lines represent the mean C Principal components from analysis of eleven environmental variables. See Table 7 for character loadings on each component.

https://binary.pensoft.net/fig/322397Table 5.

Descriptive statistics for morphometric variables of Pristimantis, subgenus Huicundomantis. Mean ± SD followed by the range of the measurements are given in each cell. ‘n’ is for the number of samples. Numbers in brackets after the species name refer to the bibliographic source of the data: (1) Lynch 1979, (2) Bustamante and Mendelson 2008, (3) Yánez-Muñoz et al. 2016, (4) Duellman and Pramuk 1999, (5) Lynch and Duellman 1995, (6) Brito et al. 2016; otherwise the source is this study. Abbreviations are: SVL = snout-vent length; HL = head length; HW = head width; TD = tympanum diameter; ED = eye diameter. All measurements are in mm.

Females

Clade

SVL

HL

HW

TD

ED

P.atillo sp. nov. n = 5

31.3 ± 2.3; 29.4–35.3

11.0 ± 0.5; 10.3–11.7

11.5 ± 0.7; 10.9–12.7

1.7 ± 0.2; 1.5–1.8

3.2 ± 0.1; 3.1–3.3

P.atratus (1) n = 10

27.4 ± 2.1; 24.9–29.2

–

–

–

–

P.balionotus (1) n = 7

28.1 ± 0.6; 27.1–29.1

–

–

–

–

P.cryptomelas (1) n = 1

38.6

–

–

–

–

P.gagliardoi (2) n = 5

30.6 ± 3.1; 26.8–33.6

10.7 ± 0.6; 9.8–11.4

12.2 ± 1.0; 10.7–13.3

1.3 ± 0.2; 1.0–1.5

3.8 ± 0.3; 3.5–4.1

P.gloria sp. nov. n = 15

30.1 ± 3.0; 26.7–35.8

11.0 ± 0.7; 10.1–12.4

11.3 ± 1.1; 9.8–13.5

1.6 ± 0.1; 1.4–1.8

3.2 ± 0.2; 2.8–3.6

P.hampatusami (3) n = 11

30.6 ± 2.1; 25.9–34.1

–

–

–

–

P.jimenezi sp. nov. n = 9

35.0 ± 2.1; 31.1–37.4

12.0 ± 0.5; 11.0–12.6

13.1 ± 0.6; 13.0–13.9

1.9 ± 0.1; 1.7–2.1

3.7 ± 0.2; 3.5–4.0

P.lutzae sp. nov. n = 15

31.4 ± 1.3; 29.7–33.9

11.3 ± 0.4; 10.7–12.1

12.2 ± 0.5; 11.4–12.9

1.7 ± 0.1; 1.5–1.8

3.4 ± 0.2; 3.1–3.8

P.multicolor sp. nov. n = 10

35.3 ± 3.5; 29.4–40.5

13.4 ± 1.1; 11.6–15.1

14.4 ± 1.3; 11.9–16.1

2.2 ± 0.2; 1.8–2.5

4.0 ± 0.3; 3.6–4.4

P.muscosus (4) n = 4

37.8; 29.6–46.1

–

–

–

–

P.nangaritza sp. nov. n = 4

29.1 ± 2.7; 25.9–32.4

11.5 ± 0.8; 10.6–12.2

11.2 ± 1.0; 10.1–12.1

1.5 ± 0.1; 1.4–1.7

3.7 ± 0.3; 3.4–4.0

P.percultus (1) n = 1

38.2

–

–

–

–

P.phillipi (5) n = 6

31.2 ± 1.1; 26.5–33.7

–

–

–

–

P.phoxocephalusn = 5

36.9 ± 2.2; 34.2–39.9

12.9 ± 0.9; 11.5–13.8

13.1 ± 1.2; 11.3–14.2

1.9 ± 0.1; 1.8–2.1

3.8 ± 0.3; 3.5–4.1

Pristimantis CCS1 n = 1

37.0

12.5

13.8

2.0

3.5

Pristimantis CCS2 n = 1

38.3

13

14.4

1.7

3.5

Pristimantis UCS1 n = 1

31.2

12.4

12.4

1.7

3.8

P.spinosus (1) n = 29

31.8 ± 1.62; 28.3–34.5

–

–

–

–

P.tinguichaca (6) n = 9

29.7 ± 1.5; 28.1–31.7

10.6 ± 0.4; 10.1–11.0

11.0 ± 0.5; 10.1–11.5

1.4 ± 0.1; 1.3–1.7

3.3 ± 0.3; 2.8–3.7

P.torresi sp. nov. n = 5

34.7 ± 3.7; 30.1–39.5

12.2 ± 1.3 10.4–13.8

13.1 ± 1.2 11.5–14.6

1.9 ± 0.2 1.8–2.2

3.6 ± 0.3 3.3–3.9

P.totoroi sp. nov. n = 7

33.0 ± 0.9; 31.9–34.3

12.1 ± 0.4; 11.5–12.6

12.3 ± 0.3; 11.8–12.6

1.6 ± 0.1; 1.6–1.8

3.3 ± 0.2; 3.1–3.6

P.verrucolatus sp. nov. n = 2

43.6 ± 4.5; 40.4–46.8

15.0 ± 1.1; 14.2–15.8

16.6 ± 1.7; 15.3–17.8

2.2 ± 0.1; 2.1–2.3

4.2 ± 0.0; 4.2–4.3

P.versicolorn = 4

27.8 ± 3.5; 24.9–32.4

10.7 ± 1.6; 9.0–12.4

10.5 ± 1.0; 9.5–11.4

1.7 ± 0.3; 1.4–2.1

3.2 ± 0.4; 2.9–3.7

Males

P.atillo sp. nov. n = 29

24.7 ± 2.5; 17.7–28.1

8.7 ± 0.7; 6.4–9.7

8.8 ± 0.9; 5.9–10.0

1.2 ± 0.1; 0.8–1.4

2.7 ± 0.3; 2.0–3.0

P.atratus (1) n = 19

21.7 ± 3.71; 17.4–24.0

–

–

–

–

P.balionotus (1) n = 2

20.0 ± 0.2; 21.8–22.2

–

–

–

–

P.chomskyi sp. nov. n = 3

28.0 ± 4.2; 24.0–32.4

9.7 ± 1.2; 8.8–11.1

10.6 ± 1.6; 9.4–12.4

1.3 ± 0.2; 1.1–1.5

3.3 ± 0.4; 2.9–3.6

P.cryptomelas (1) n = 4

29.2; 28.2–30.3

–

–

–

–

P.gagliardoi (2) n = 5

22.2 ± 2.0; 19.1–24.3

7.9 ± 0.9; 6.8–9.1

9.0 ± 0.9; 7.7–10.2

0.8 ± 0.0; 0.7–0.8

2.8 ± 0.2; 2.4–3.0

P.gloria sp. nov. n = 24

21.7 ± 2.3; 16.8–24.7

8.6 ± 0.7; 6.7–9.4

8.4 ± 0.8; 6.5–9.5

1.2 ± 0.1; 0.9–1.4

2.6 ± 0.2; 2.1–2.9

P.hampatusami (3) n = 28

21.0 ± 1.8; 17.0–24.9

–

–

–

–

P.jimenezi sp. nov. n = 12

25.5 ± 1.6; 21.8–27.1

8.9 ± 0.6; 7.8–9.6

9.1 ± 0.6; 7.8–9.7

1.4 ± 0.1; 1.3–1.5

3.06 ± 0.24; 2.56–3.37

P.lutzae sp. nov. n = 14

24.6 ± 1.7; 21.4–27.0

8.7 ± 0.5; 7.9–9.4

9.3 ± 0.7; 8.2–10.4

1.3 ± 0.1; 1.1–1.4

2.77 ± 0.19 2.53–3.26

P.multicolor sp. nov. n = 12

26.2 ± 3.5; 19.7–29.7

9.6 ± 1.0; 8.0–11.0

10.1 ± 1.2; 8.0–11.5

1.5 ± 1.2; 1.1–1.8

3.18 ± 0.49; 2.45–3.82

P.nangaritza sp. nov. n = 13

18.8 ± 1.1; 17.4–20.8

7.6 ± 0.6; 7.1–8.8

7.2 ± 0.5; 6.6–8.3

1.0 ± 0.1; 0.8–1.2

2.69 ± 0.17 2.49–3.08

P.percultus (1) n = 1

29.8

–

–

–

–

P.phillipi (5) n = 10

23.0 ± 0.4; 21.1–25.1

–

–

–

–

P.phoxocephalusn = 3

24.7 ± 3.2; 20.8–27.9

9.0 ± 1.36; 7.4–10.6

8.7 ± 1.5; 7.0–10.4

1.2 ± 0.2; 1.0–1.3

2.66 ± 0.25 2.36–2.90

Pristimantis UCS2 n = 2

20.5 ± 1.7; 19.3–21.7

7.6 ± 0.7; 7.1–8.1

7.3 ± 0.4; 7.0–7.6

1.0 ± 0.1; 0.9–1.0

2.43 ± 0.23 2.29–2.62

P.spinosus (1) n = 34

20.1 ± 1.8; 16.1–25.0

–

–

–

–

P.teslai sp. nov. n = 4

25.2 ± 1.8; 23.4–27.3

8.9 ± 0.4; 8.4–9.2

9.0 ± 0.6; 8.2–9.5

1.3 ± 0.1; 1.2–1.5

2.89 ± 0.14 2.76–3.07

P.tinguichaca (6) n = 11

23.4 ± 1.1; 21.2–24.7

8.7 ± 0.3; 8.2–9.2

8.6 ± 0.5; 7.9–9.4

1.3 ± 0.2; 1.1–1.6

2.8 ± 0.4 2.3–3.3

P.torresi sp. nov. n = 18

25.9 ± 2.1; 23.3–30.0

9.2 ± 0.6; 8.4–10.5

9.3 ± 0.7; 8.4–10.5

1.3 ± 0.1; 1.2–1.6

2.98 ± 0.23 2.64–3.47

P.totoroi sp. nov. n = 21

26.8 ± 2.0; 23.2–29.4

9.6 ± 0.6; 8.5–10.5

9.5 ± 0.6; 8.4–10.3

1.3 ± 0.1; 1.1–1.4

2.79 ± 0.25 2.27–3.25

P.verrucolatus sp. nov. n = 15

29.4 ± 2.7; 25.1–34.5

9.7 ± 0.7; 8.5–10.7

10.5 ± 1.0; 8.5–12.1

1.5 ± 0.1; 1.3–1.8

3.21 ± 0.24 2.79–3.64

P.versicolorn = 12

20.5 ± 1.6; 18.1–23.3

8.2 ± 0.5; 7.3–8.9

7.6 ± 0.6; 6.7–8.6

1.3 ± 0.2; 1.0–1.6

2.84 ± 0.32 2.32–3.36

Bioacoustic analyses

Bioacoustic data are summarized in Table 6. The available advertisement calls of P.phoxocephalus and three candidate species have a similar structure: one to several whistles (Fig. 6). Calls from Cashca Totoras (Bolívar Province, Ecuador) were distinct from those from Pilaló (Cotopaxi Province, Ecuador) even though both populations had historically been ascribed to P.phoxocephalus. The clade from high altitudes of Azuay Province shows, by far, the longest duration of the note, the lowest dominant frequency, and the highest variation between the initial and final frequency of the note. The advertisement call of the clade from lower altitudes of Azuay shows the highest frequency of the first and second harmonic. Pristimantisphoxocephalus shows the shortest interval between notes. All analyzed species are separated by large genetic distances (>5.6%). They also have unequivocally distinct advertisement calls (Fig. 6).

Descriptive statistics for bioacoustic variables of Pristimantisjimenezi sp. nov., P.phoxocephalus, P.totoroi sp. nov. and P.verrucolatus sp. nov. Mean ± SD and range in brackets are given in each cell. The number of samples is given in brackets after species name.

P.jimenezi (2)

P.phoxocephalus (2)

P.totoroi (2)

P.verrucolatus (2)

Notes per call

1–2

3–9

2–6

1

No. harmonics

4

4–5

3–7

4–7

Note duration (s)

0.182 ± 0.035

0.154 ± 0.030

0.125 ± 0.038

0.433 ± 0.037

(0.157–0.206)

(0.133–0.175)

(0.098–0.152)

(0.407–0.459)

Interval between notes (s)

0.297 ± 0.088

0.135 ± 0.035

0.339 ± 0.038

NA

(0.234–0.360)

(0.110–0.159)

(0.312–0.366)

Peak time (s)

0.091 ± 0.018

0.077 ± 0.014

0.063 ± 0.019

0.216 ± 0.018

(0.080–0.103)

(0.067–0.087)

(0.049–0.076)

(0.204–0.230)

Dominant frequency (Hz)

2894.58 ± 167.49

2578.12 ± 0

2569.65 ± 21.21

2114.08 ± 265.17

(2776.15–3013.01)

(2512.22–2627.07)

(1926.58–2301.58)

Initial frequency (Hz)

2789.89 ± 232.02

2452.15 ± 87.01

2550.96 ± 148.20

1837.5 ± 212.13

(2625–2953.13)

(2390.63–2513.67)

(2446.16–2655.75)

(1687.5–1987.5)

Final frequency (Hz)

2976.56 ± 232.02

2586.91 ± 12.43

2619.88 ± 50.73

2231.25 ± 238.65

(2812.5–3140.63)

(2578.13–2595.70)

(2584–2655.75)

(2062.5–2400)

Frequency change (Hz)

187.5 ± 0

134.77 ± 99.44

68.92 ± 97.47

393.75 ± 26.52

(64.45–205.08)

(0–137.84)

(375–412.5)

2nd harmonic frequency (Hz)

5373.79 ± 412.25

4532.23 ± 62.15

4714.43 ± 453.59

4171.89 ± 596.64

(5446.29–6029.3)

(4488.28–4576.17)

(4393.69–5035.16)

(3750–4593.78)

Environmental analyses

In the PCA, the eleven environmental variables were reduced to four principal components (PCs) with an eigenvalue >1. Together they accounted for 81.58% of the variation. The highest loadings for each PC were: temperature-associated variables for PC I, vegetation-associated variables and precipitation of the coldest quarter for PC II, precipitation-associated variables for PC III, and NDVI and temperature seasonality for PC IV. Percentage of explained variance and variable loadings for each PC are shown in Table 7.

Character loadings, eigenvalues and percentage of explained variance for Principal Components (PC) I–IV. The analysis was based in eleven environmental variables extracted from presence localities of 26 species of Pristimantis, subgenus Huicundomantis. Bold figures indicate highest loadings.

Variable

PCI

PCII

PCIII

PCIV

Annual mean temperature

0.956

0.014

-0.139

0.153

Mean diurnal temperature range

0.680

-0.365

-0.026

-0.383

Temperature seasonality

-0.257

0.016

0.465

0.642

Max temperature of warmest month

0.966

-0.046

-0.122

0.121

Min temperature of coldest month

0.930

0.050

-0.153

0.201

Annual precipitation

0.382

-0.395

0.777

-0.101

Precipitation of warmest quarter

0.219

0.156

0.709

0.234

Precipitation of coldest quarter

0.428

-0.605

0.210

-0.268

Gross primary production

0.377

0.839

0.128

-0.185

Leaf area index

0.469

0.797

0.146

-0.125

Normalized difference vegetation index

0.339

-0.229

-0.378

0.611

Eigenvalue

4.096

2.074

1.606

1.198

Cumulative variance (%)

37.23

56.09

70.69

81.58

Integrative analyses

By combining evidence from the abovementioned character sets, we conclude that our study group is composed of 28 candidate species. Of them, 25 are confirmed candidate species and 3 are unconfirmed. Of the CCS, 12 have available names and the 13 remaining are new species. We describe 11 of them below. We did not describe two CCS because we did not have enough specimens to characterize species variation. Because of their similar morphology to the examined species, and after a thorough bibliographic analysis, we propose P.balionotus (Lynch, 1979) and P.percultus (Lynch, 1979) as part of our clade of study.

We identified that the recently described P.hampatusamiYánez-Muñoz et al., 2016 and P.prometeiiSzékely et al., 2016 belong to the same species. We compared specimens of both species from the QCAZ and INABIO collections. Our comparisons showed that both species are morphologically indistinguishable. The type locality for both species is the same further suggesting that both represent a single species. With this evidence, we propose P.prometeii as a junior synonym for P.hampatusami given the principle of priority.

We found a misidentification of two Genbank sequences with accession numbers KU217863 and KU218025. GenBank identifications are P.cryophilius and P.phoxocephalus, respectively. They actually are P.philipi (KU217863) and P.totoroi sp. nov. (KU218025). Updated identifications of sequences used in this study are shown in Table 1.

In the following section, we present the descriptions of the new species and new taxa identified in this study. We also include an updated diagnosis of P.phoxocephalus sensu stricto, as diagnoses made in its description and later revisions (e.g., Duellman and Pramuk 1993; Lynch and Duellman 1997) included information of non-conspecific populations.

This clade is strongly supported by genetic evidence (Fig. 3). Morphological synapomorphies are unknown. Members of this clade are characterized by: (i) dorsolateral folds absent (except for P.atratus); (ii) cranial crests absent (except for P.atratus, P.percultus, and P.spinosus); (iii) tympanic membrane and tympanic annulus prominent (both absent in P.philipi); (iv) dentigerous processes of vomer present; (v) small to prominent tubercles on heel; (vi) fingers with lateral fringes; (vii) basal webbing between toes (except for P.balionotus, P.hampatusami, and P.philipi which lack webbing); (viii) Toe V longer or much longer than Toe III (Figs 7–9); (ix) in life, groins and concealed surfaces of thighs with distinctive coloration patterns including flash colors and light or bright colored flecks or spots on a darker background; colors, shapes and sizes of these ornaments are variable among species; (x) SVL females 24.9–46.8 mm; SVL males 16.1–34.5 mm. Species of this clade may bear a fleshy keel or a papilla at the tip of the snout, and lateral, middorsal, or postocular folds.

Huicundomantis occurs in Eastern and Western Andean slopes and Inter-Andean valleys of southern and central Ecuador, and Eastern Andean slopes of northern Peru. They inhabit the following Natural Regions: Deciduous Costa Forest, Western Foothill Forest, Western Montane Forest, Paramo, Inter-Andean Shrub, Eastern Montane Forest, and Eastern Foothill Forest, between elevations of 230 and 4200 m a.s.l.

Etymology.

We name this clade Huicundomantis because these frogs are frequently found inside bromeliad plants. Huicundo is a word in Quechua, an indigenous South American language, locally used to referring to bromeliads.

Pristimantisphoxocephalus species group new taxon

Definition. The P.phoxocephalus species group is strongly supported in our phylogeny. Members of this group share the following morphological traits: (i) dorsolateral folds absent (except for P.atratus); (ii) snout with a fleshy keel or papilla at the tip; (iii) cranial crests absent (except for P.atratus and P.percultus); (iv) tympanic membrane and tympanic annulus prominent; (v) dentigerous processes of vomer present; (vi) males with vocal slits (except for P.versicolor); (vii) fingers and toes with lateral fringes; (viii) basal webbing between toes (except for P.balionotus and P.hampatusami); (ix) Toe V longer or much longer than Toe III (Fig. 7); (x) in life, groins and concealed surfaces of thighs with distinctive coloration, including flash colors and light or bright colored flecks or spots on a darker background; colors, shapes, and sizes of ornaments variable among species; (xi) SVL females 24.9–46.8 mm; SVL males 16.8–34.5 mm.

Holotypes of Pristimantisatillo sp. nov. and P.chomskyi sp. nov. Photographs of preserved holotypes of AP.atillo (QCAZ 42500, male) and BP.chompskyi (QCAZ 47515, male). Dorsal view on the left, ventral view on the right. Specimens are shown at the same scale.

Pristimantisatillo is similar to other species with acuminate and protruding snouts of this group such as P.jimenezi sp. nov., P.phoxocephalus, P.teslai sp. nov., P.torresi sp. nov., P.totoroi sp. nov., and P.verrucolatus sp. nov. The bright orange coloration of its groins and posterior surfaces of thighs distinguishes it from them (brown with small light brown to yellow spots in P.jimenezi sp. nov.; yellow with black reticulations in P.phoxocephalus; dark brown with yellow irregular blotches in P.teslai sp. nov.; brown with or without yellow spots in P.torresi sp. nov.; reddish brown with small light brown to yellow spots in P.verrucolatus sp. nov.). Pristimantisatillo can be further distinguished from P.jimenezi sp. nov. and P.phoxocephalus by the presence of its characteristic black dots in the flanks (black dots absent in P.jimenezi sp. nov. and P.phoxocephalus). Pristimantisatillo differs from P.teslai sp. nov. by the dorsal skin texture (shagreen in P.atillo; tuberculate in P.teslai sp. nov.). The copper coloration of the iris differentiates P.atillo from P.torresi sp. nov. and P.totoroi sp. nov., whose iris is golden with a red streak. Furthermore, tubercles and folds of P.totoroi sp. nov. are more prominent than those of P.atillo, and its head, relative to the body, is longer (males Wilcoxon’s Z = 4.50124, p < 0.001, HL/SVL = 33.5–38.5% in P.atillo, 34.2–38.7% in P.totoroi sp. nov.; females Z = 2.43599, p = 0.0149, HL/SVL = 33.1–36.6% in P.atillo, 35.2–37.6% in P.totoroi sp. nov.). Pristimantisverrucolatus sp. nov. differs from P.atillo in having large tubercles and warts on its flanks. Pristimantisatillo is similar to P.modipeplus (Lynch 1981) in coloration pattern; P.modipeplus has a subacuminate snout in dorsal view and rounded in profile, lacks basal webbing between toes, and large females bear low cranial crests (Lynch 1981); meanwhile, P.atillo has an acuminate and protruding snout with a keel at the tip, presents basal webbing between toes and has no cranial crests.

This section is based on 45 preserved specimens of the type series and photographs available for 11 individuals. Variation in living and preserved individuals is shown in Figures 11, 12. Coloration in life is mentioned in parenthesis. Dorsum varies from pale to dark brown with or without lighter irregular marks or white spots scattered on dorsum, few individuals have several thin parallel longitudinal stripes or a middorsal band (i.e., longitudinal pattern); the interorbital band can be thin to broad, paler or darker than background and is absent in individuals with longitudinal pattern; interscapular blotch is often present. Flanks have pale oblique reticulations usually surrounded by dark stripes formed by rows of black flecks. Groins, anterior and posterior surfaces of thighs cream (orange, in most cases surrounded by yellow blotches; an individual has yellow groins (QCAZ 42502)). Dorsal surfaces of thighs with brown to black oblique stripes. Venter and throat coloration varies from cream to white (cream to white with different levels of transparency, with or without ill-defined yellow blotches) with or without black flecks. Iris is reddish to orangey copper with a faint medial horizontal darker streak and thin black reticulations; sclera varies from white to light blue.

This species is only known from the type locality, surroundings of Lagunas de Atillo, and nearby locations at Sangay National Park, between 3185 and 3730 m a.s.l (Fig. 1). This corresponds to the Eastern Montane Forest and Paramo biogeographic regions. Most individuals collected at night were found active on low vegetation, from ground level up to 1 m above the ground; an individual was collected from a bromeliad 2.5 m above the ground. During the day, they were found beneath rocks. Most collections were made on roadsides. Calling males have been found in March.

According to available data, this species has a very restricted distribution (Extent of Occurrence 7 km2, Area of Occupancy 16 km2). However, less accessible adjacent areas in Sangay National Park are unexplored and represent potential distribution areas for this species. Therefore, we consider this species to be Data Deficient (IUCN 2017).

Etymology.

The specific epithet refers to the type locality of this species, the surroundings of Lagunas de Atillo, a lake complex in Sangay National Park, a UNESCO World Heritage Site.

Based on the 12 preserved specimens of the type series and photographs from eight individuals. Variation in life and preservative is shown in Figures 13, 14. Coloration in life is given in parenthesis. Dorsum and flank coloration vary from brown to brownish cream (dark brown to orange). Dorsal markings may be absent or present as longitudinal stripes or dark blotches evenly scattered; one individual has a pale blotch on dorsum. All individuals have brown supratympanic and canthal stripes; some also have dark interorbital stripe and labial bars. Color of groins, armpits, dorsal surfaces of thighs, concealed surfaces of thighs, shanks and tarsus is dark brown (dark chocolate brown, suffused or not with orange, with or without small cream flecks), few individuals with small pale flecks; dorsal surfaces of thighs and groins can have irregular pale spots. Limbs bear scattered small dark spots arranged or not as transversal bands. Venter varies from cream to dusty brown with or without dark markings (orange, cream, or dusty brown, with or without darker flecks or mottling). The iris is orange with a faint red medial streak and thin black reticulations. Sclera varies from white to light blue.

Distribution, natural history, and conservation status.

This species is only known from the type locality, Tapichalaca Reserve, Zamora Chinchipe Province, Ecuador at 3366 m a.s.l (Fig. 1), which corresponds to the Paramo biogeographic region. All individuals were found at night inside bromeliads, mostly of the genus Puya. Tapichalaca Reserve is adjacent to Podocarpus National Park (northward) and Yacuri National Park (southward). Both areas are unexplored and represent potential habitats for this species. Therefore, we assign P.chomskyi to the Data Deficient Red List Category (IUCN 2017).

Etymology.

The specific epithet is a noun in the genitive case and is a patronym for Noam Chomsky, US born theoretical linguist and one of the most cited modern scholars. Chomsky is the founder of modern linguistics. He developed the concept of “universal grammar,” an innate cognitive capacity, shared by all humans, which allows to learn and communicate through complex speech. Chomsky was professor at the Massachusetts Institute of Technology between 1950 and 2017. He is currently professor at Arizona State University.

Holotypes of Pristimantisgloria sp. nov. and P.jimenezi sp. nov. Photographs of preserved holotypes of AP.gloria (QCAZ 57201, female) and BP.jimenezi (QCAZ 45170, male). Dorsal view on the left, ventral view on the right. Specimens are shown at the same scale.

Similar species are P.balionotus, P.chomskyi, P.lutzae sp. nov., P.multicolor sp. nov., P.percultus, and Pristimantis sp. (CCS2). Pristimantisgloria is readily distinguished from them (except P.percultus) by the presence of wide black reticulations in its iris (thin black reticulations in the other species). Pristimantisgloria can be further distinguished from P.balionotus by having basal webbing between toes (absent in P.balionotus) and warty flanks (tuberculate in P.balionotus). Pristimantischomskyi is different from P.gloria by having shagreen dorsal skin (tuberculate to warty in P.gloria), lacking a middorsal fold (present in P.gloria), having a larger body (males Z = -2.31490, p = 0.0206, SVL = 23.98–32.38 mm in P.chomskyi, 16.7–24.74 mm in P.gloria), a smaller tympanum (males Z = 2.66173, p = 0.0078, TD/SVL = 4.5–4.7% in P.chomskyi, 5.1–6.2% in P.gloria) and larger eye (males Z = -2.50743, p = 0.0122, ED/SVL = 11.1–12% in P.chomskyi, 11–13.8% in P.gloria sp. nov.), relative to body length. The dorsum and flanks of Pristimantislutzae sp. nov. are predominantly covered with tubercles, while it is mostly covered with warts in P.gloria; the ratio between the length and width of the head is larger in P.gloria than P.lutzae sp. nov. (males Z = -5.00826, p < 0.0001, HL/HW = 96.8–114.5% in P.gloria, 90–97% in P.lutzae sp. nov.; females Z = -3.77517, p = 0.0002, HL/HW = 92–105% in P.gloria, 90–96% in P.lutzae sp. nov.). Morphometrically, P.multicolor sp. nov. is larger than P.gloria (males Z = 3.07054, p = 0.0021, SVL 16.7–24.7 mm in P.gloria, 19.7–29.7 mm in P.multicolor sp. nov.; females Z = 2.85671, p = 0.0043, SVL 26.7–35.8 mm in P.gloria, 29.3–40.5 mm in P.multicolor sp. nov.), and has a wider head (males Z = 2.23159, p = 0.0256, HW/SVL = 36.1–40.7% in P.gloria, 36.3–40.9% in P.multicolor sp. nov.; females Z = 4.13252, p < 0.0001, HW/SVL = 36.1–39% in P.gloria, 39.6–42.2% in P.multicolor sp. nov.) relative to body length; furthermore, P.multicolor sp. nov. lacks a middorsal fold. Pristimantispercultus, like P.gloria, has wide black reticulations on the iris, and warts and tubercles on flanks. They can be readily recognized because P.percultus has a keel on the snout (absent in P.gloria), cranial crests (absent in P.gloria), and is larger (Table 5). Pristimantis sp. (CCS2) is the closest species to P.gloria (Fig. 3, Table 2). CCS2 has a red dorsum, a golden iris with a red medial streak and thin reticulations, and broadly expanded discs on finger (Fig. 4M); while the dorsum of P.gloria is light to dark brown, its iris is cream to light silver with a red medial streak and wide black reticulations. Though there is only one female available for CCS2, it has a larger body and smaller tympanum than any individual of P.gloria (Table 5). Pristimantisgloria is also similar to the Peruvian species Pristimantispataikos (Duellman & Pramuk, 1999) from which can be distinguished by the presence of nuptial pads in adult males (absent in P.pataikos), the presence of lateral fringes on fingers and toes (absent in P.pataikos), the presence of vomerine odontophores (absent in P.pataikos), and the presence of pale spots on the groins and concealed surfaces of thighs (uniformly brown in P.pataikos) (Duellman and Pramuk 1999).

This section is based in 60 individuals of the type series and photographs available from 15 individuals. Variation in life and preservative is shown in Figures 16, 17. In preservative, dorsal coloration varies from light to dark brown with or without shades of gray. Dorsum uniformly colored or bearing a pattern of parallel longitudinal stripes, irregular reticulations or pale blotches. Most individuals have scattered black spots on dorsum, especially on dorsolateral surfaces; three individuals (QCAZ 57201, QCAZ 26369, QCAZ30741) display scattered white spots. Black or brown canthal bars, supratympanic stripes, labial bars, and interorbital bars or stripes can be present in this species. Flanks have the same or slightly lighter hue than dorsum. Groins and posterior surfaces of thighs bear pale irregularly shaped small spots or flecks on a brown background; when flecking is very dense the background color resembles brown reticulations. One individual lacks these flecks and spots (QCAZ62778). Limbs bear or not transversal bars and black spots. Ventral coloration varies from cream to dirty cream. In life, dorsal coloration varies from light to dark brown with or without shades of cream, yellow or orange. When present, reticulations and longitudinal stripes are brighter colored than the rest of dorsum and bordered by thin black stripes. Dorsum usually bears black spots, rarely, white spots. When present, canthal and supratympanic bars or stripes are brown or black, interorbital bars or stripes are black, brown or yellow. Flanks are the same color as dorsum or lighter. Groins and posterior surfaces of thighs are brown, creamish brown, pinkish brown or purplish brown with irregular flecks or spots; when flecking is dense, the background color resembles thin reticulations (QCAZ 62778 is the only individual that do not present these spots). Venter varies from cream to brownish cream. Iris is cream or light silver with wide black reticulations that can cover almost completely the iris; a faint red to reddish brown medial streak is present (Fig. 16D); sclera varies from cream to light blue.

Pristimantisgloria is known from Inter-Andean Shrub and Paramo regions in Azuay, Loja, and Morona Santiago Provinces, between 2460 and 3525 m a.s.l. (Fig. 1). Most individuals were found underneath rocks in paramos or pastures near the roadside, during day or night. Two couples in amplexus were found underneath rocks at night in April. A calling male was found in low vegetation at 19h00 on October 16, 2013.

We consider P.gloria as an Endangered species following B1ab(iii) + 2ab(iii) IUCN criteria because: (i) it is only known from five localities (sensu IUCN 2017) in non-protected areas, partly affected by human settlements, agriculture and cattle raising; (ii) its Extent of Occurrence is estimated to be less than 5000 km2 (617 km2); and (iii) its Area of Occupancy < 500 km2 (44 km2).

Etymology.

The specific epithet is a patronym for Gloria Lorena Rosales Narváez and Gloria María Esmeralda Narváez, mother and grandmother of the leading author. This species is named after them in gratitude for all their love and support.

Remarks.

Because of the previous lack of molecular data, this species has been mistakenly identified as P.riveti (e.g., collections at the QCAZ museum). Here, we recognize it as a different species and assign it to the P.phoxocephalus species group. It has the lowest genetic divergence with its closest species, Pristimantis sp. CCS2; the average uncorrected p distance between them is 2%. The differences in morphology between them are so evident that we consider 2.0% as our reference value to delimit candidate species.

Pristimantisjimenezi is similar to P.atillo, P.phoxocephalus, P.teslai sp. nov., P.torresi sp. nov., P.totoroi sp. nov., and P.verrucolatus sp. nov. It differs from P.atillo in the coloration of the groins and posterior surfaces of thighs (orange in P.atillo; pinkish to dark brown with small light brown to yellow spots in P.jimenezi), and flanks (black dots and flecks surrounding light reticulations in P.atillo; lacking black dots in P.jimenezi). Pristimantisjimenezi can be distinguished from P.phoxocephalus by its groin coloration (yellow with black reticulations in P.phoxocephalus) and advertisement call. The call of P.phoxocephalus has shorter inter-note intervals and a lower frequency of the second harmonic and final frequency of the note (Table 6). Pristimantisteslai sp. nov. has tuberculate dorsal skin (shagreen in P.jimenezi) and lacks lateral folds, which are present in P.jimenezi. The most similar species to P.jimenezi is P.torresi sp. nov., which has golden to beige iris with a red medial streak (copper to red in P.jimenezi). P.totoroi sp. nov. has more prominent folds and tubercles than P.jimenezi, and its head length is larger (males Z = 3.05006, p = 0.0023, HL/SVL = 33.4–37.5% in P.jimenezi, 34.2–38.7% in P.totoroi sp. nov.; females Z = 2.85798, p = 0.0043, HL/SVL = 32.8–37.3% in P.jimenezi, 35.2–37.6% in P.totoroi sp. nov.), its tympanum smaller (males Z = -4.24763, p < 0.0001, TD/SVL = 4.8–6% in P.jimenezi, 4.4–5.1% in P.totoroi sp. nov.; females Z = -2,96383, p = 0.003, TD/SVL = 5–5.6% in P.jimenezi, 4.8–5.3% in P.totoroi sp. nov.), and its eyes smaller (males Z = -4.06051, p < 0.001, ED/SVL = 11.1–13.3% in P.jimenezi, 9.2–11.6% in P.totoroi sp. nov.; females Z = -2.43458, p = 0.0149, ED/SVL = 9.5–11.8% in P.jimenezi, 9.5–10.6% in P.totoroi sp. nov.) than those of P.jimenezi. Furthermore, advertisement calls of P.totoroi sp. nov. have shorter notes and lower dominant, second harmonic, and final frequencies of the notes than those of P.jimenezi (Table 6). Pristimantisverrucolatus sp. nov., which has an adjacent distribution to P.jimenezi at high elevations, can be distinguished by the presence of large tubercles and warts on flanks (absent in P.jimenezi) and by its smaller size (males Z = 3.58643, p = 0.0003, SVL = 21.8–27.0 mm in P.jimenezi, 25.1–34.5 mm in P.verrucolatus sp. nov.; females Z = 2.00347, p = 0.0451, SVL = 31.1–37.4 mm in P.jimenezi, 40.4–46.8 mm in P.verrucolatus sp. nov.). Additionally, they can be distinguished by their advertisement calls: the call of P.verrucolatus sp. nov. consists of a single note, longer than the one or two notes of the call of P.jimenezi; the dominant frequency is higher in advertisement calls of P.jimenezi, and the change in frequency along the note is lower in P.jimenezi (Table 6).

Data of preserved individuals is based on 25 specimens of the type series; information of coloration in life is based on photographs from nine individuals. Variation of preserved and live individuals is shown in Figures 18, 19. Coloration in life is provided in parenthesis. Dorsum is creamy to light and dark brown, with or without lighter irregular marks or chevrons; some individuals show a middorsal band; a pale W-shaped mark on scapula is often present; head bears an interorbital stripe or band paler or darker than dorsum; most individuals bear labial bars, and a few, canthal bars (black to brown). Flanks often bear pale (light-brown to yellow) spots or reticulations all over them or confined to the groins and axils. Groins and posterior surfaces of thighs cream with small pale spots (salmon, pinkish brown, purplish, purplish brown or dark brown with small light-brown to yellow spots). Dorsal surfaces of thighs are darker than dorsum bearing pale (yellow or light brown) reticulations. Ventral surfaces of the body are cream (yellowish to pale cream) with or without dark flecks or reticulations on the belly and throat. Iris varies from copper with a red medial streak of variable size to completely red; it bears thin black reticulations; white to light-blue sclera.

Based on recordings of QCAZ 45178 (January 20, 2010; 19h10; 12 °C) and QCAZ 46977 (August 23, 2009; 16h30). The advertisement call of P.jimenezi consists of one or two peep-like notes (Fig. 6A). The average duration of a note is 0.18 s (range 0.016–0.21 s), and the interval between them is 0.30 s (range 0.23–0.36 s). The fundamental and dominant frequencies of the notes are on average 2894 Hz (range 2776–3013 Hz). The peak time of the note occurs exactly in the middle of its duration. There is an increase in frequency along the note (2790–2977 Hz on average). Descriptive statistics for bioacoustic parameters are shown in Table 6.

Distribution, natural history, and conservation status.

This species is known from Western Andean slopes of Azuay Province between elevations of 1800 and 2900 m (Fig. 1); this corresponds to the Western Montane Forest region. Individuals of this species inhabit primary and secondary forest. Specimens were found at afternoon and night on low vegetation up to 1.5 m above the ground and inside bromeliads up to 8 m above the ground. Some individuals were collected from banana plants. Gravid females and calling males were collected in January and August; males were found calling at 16 and 19 h.

Following B1ab(iii) IUCN criteria, we consider P.jimenezi to be Critically Endangered. Available records come from two localities (sensu IUCN 2017) where suitable surrounding habitats are disturbed by human settlements, agriculture, and cattle raising. Its Extent of Occurrence is estimated to be less than 100 km2 (39 km2).

Etymology.

The specific epithet is a noun in the genitive case and is a patronym for Marcos Jiménez de la Espada, a Spanish naturalist who visited South America between 1862 and 1865. During his trip, he made significant collections of amphibians including new species that he described between 1870 and 1875. Among others, he named the genus Pristimantis and 19 species of Ecuadorian amphibians. His species descriptions are remarkably detailed in comparison with those of his contemporaries. He also made accurate and detailed descriptions of the natural history and the external and internal morphology of Neotropical anurans.

Variation in preservative is based on 40 individuals of the type series and photographs from eight individuals. Variation in life and preservative is shown in Figures 21, 22. Coloration in life is provided in parenthesis. Dorsal coloration varies from light to dark gray or brown (light, orangey or dark brown); markings on dorsum (light brown, yellow or orange) are present or absent, most individuals have irregular reticulations, some have a series of parallel longitudinal stripes; dorsum and flanks may bear scattered black or white spots. All individuals bear supratympanic and canthal stripes (black, brown); an interorbital stripe or band is present except for individuals with the longitudinal pattern on dorsum. Flanks have the same background color as dorsum. Groins, posterior and anterior surfaces of thighs are cream to brown with small paler spots (pinkish, purplish or reddish brown, suffused or not with orange, with cream or light brown spots). Limbs bear dark transversal bands or scattered small dark spots. Ventral coloration varies from cream to dusty cream (white to cream); venter with or without brown reticulations. The iris is golden to creamy brown with a reddish dark brown medial horizontal streak. White to light-blue sclera.

Pristimantislutzae is known from Paramo, Inter-Andean Shrub, Western and Eastern Montane Forest in the Andes of Azuay and Cañar Provinces in Ecuador, between 2895–4100 m a.s.l (Fig. 1). Individuals collected at night were found in bunch grasses, pastures, and low vegetation up to 80 cm above the ground. Individuals collected at day were found in pastures or underneath rocks. Calling males have been found on bunch grasses or low vegetation during February, July, and December at night.

Despite the relatively small distribution range of this species (Extent of Occurrence = 2338 km2) we assign it to the Least Concern Red List category because its distribution overlaps with four protected areas, Cajas National Park, Mazán Reserve, Mazar Wildlife Reserve, and Yanuncay Irquis Protected Forest, and it is a common species in these places.

Etymology.

The specific epithet is a noun in the genitive case and is a patronym for Bertha Lutz, who was a Brazilian herpetologist. We name this species after her in recognition of her scientific career and her activism in the fight for gender equality.

Remarks.

Specimens of this species were previously referred as Pristimantisriveti (Despax 2011) based on Lynch (1979) characterization of the species (e.g., Almendáriz and Orcés 2006; Heinicke et al. 2007; Padial et al. 2014). Photographs of the holotype of P.riveti (Fig. 23B) as well as the location of its type locality indicate that P.lutzae is a different species. See Taxonomic status of P.riveti section for details. Through morphological and molecular evidence, we recognize P.lutzae as a different species and assign it to the P.phoxocephalus species group.

Based on 60 preserved specimens and photographs from 57 individuals. Variation of live and preserved individuals is shown in Figures 24, 25. Texture of skin varies among individuals. Dorsum varies from shagreen to warty, with very low warts (not tuberculate); flanks vary from tuberculate to warty, warts on flanks are larger than those of dorsum; tubercles on ulna, shanks, and tarsus can be distinct or inconspicuous. Coloration in life is highly variable (Fig. 24). Dorsal surfaces can be yellow, cream, orange, or pale to dark brown with or without olive or orange tones. They can have few to abundant black spots on dorsum, flanks, and dorsal surfaces of limbs. Dorsum occasionally bears pale scapular or sacral blotches, a middorsal stripe, or white spots. Head usually bears black or brown canthal and supratympanic stripes, sometimes an interorbital bar. Groins and posterior surfaces of thighs are cream, orange, reddish brown, orangey brown, brown, dark brown, or black with or without cream to yellow spots. The background color of the venter varies from cream to dark brown with different levels of transparency; small beige to yellow blotches can be present on the belly, chest, and throat, but in some individuals are restricted to the chest. Iris is copper, golden, creamy yellow or creamy brown with or without a dark brown to red medial horizontal streak; black reticulations are present; sclera can be light brown, cream, white, or light blue.

Pristimantismulticolor is known from Parque Nacional Yacuri, near the border with Peru, at Loja and Zamora Chinchipe Provinces (Fig. 1). It inhabits Paramo and Eastern Montane Forest between 3186 and 3515 m. During the late afternoon and night, individuals were commonly found beneath or over moss, mat-forming plants, tussock grasses and rocks, perched on branches and leaves of shrubs up to 1.75 m above ground, or inside bromeliads, terrestrial or epiphytes, up to 1 m above the ground. During the day they were found inside bromeliads. Calling males and gravid females were found in December, April, and May; males were calling on low branches of shrubs.

According to available data, P.multicolor has a very restricted distribution (Extent of Occurrence = 10 km2) but is locally abundant. However, we propose assigning it to the Data Deficient Red List Category (IUCN 2017) because extensive areas in Yacuri National Park remain unexplored and represent potential distribution for this species.

Etymology.

The specific epithet comes from the Latin words multus meaning many, and color meaning color. It refers to the wide range of color variation in this species (Fig. 24).

Remarks.

This species was collected for the first time in 2015 by personnel from the QCAZ museum during expeditions to Yacuri National Park. It was identified as Pristimantisaff.riveti. Here, we recognize it as a different species and assign it to the P.phoxocephalus species group.

Pristimantisphoxocephalus is most similar to P.atillo, P.jimenezi, P.teslai sp. nov., P.totoroi sp. nov., and P.verrucolatus sp. nov. However, the coloration of the groins and concealed surfaces of thighs (yellow coloration with dark brown to black reticulations) distinguishes it from the species above (orange in P.atillo; different shades of brown with light brown to yellow flecks, spots or blotches in the other species). It can be further distinguished from P.jimenezi by having an advertisement call with shorter inter-note interval and a lower frequency of the second harmonic and final frequency of the note (Table 6). Pristimantisteslai sp. nov. has a tuberculate dorsum while in P.phoxocephalus is shagreen. Pristimantistorresi sp. nov. differs from P.phoxocephalus in having a golden to beige iris with a red to reddish-brown medial streak (copper with or without a faint red streak in P.phoxocephalus), and a wider head relative to its body (males Z = -2.56285, p = 0.0104, HW/SVL = 33.4–34.4% in P.phoxocephalus, 34.2–37.8% in P.torresi sp. nov.; females Z = -2.08893, p = 0.0367, HW/SVL = 33.1–37.3% in P.phoxocephalus, 36.7–38.5% in P.torresi sp. nov.). Pristimantistotoroi sp. nov. is the most similar and geographically closest species; both species differ by the coloration of the groins and iris (copper with or without a brown streak in P.phoxocephalus; golden with a red streak in P.totoroi sp. nov.) and by the presence of more distinctive tubercles and dermal folds in P.totoroi sp. nov. The advertisement call of P.totoroi sp. nov. has shorter inter-note intervals than P.phoxocephalus (Table 6). Pristimantisverrucolatus sp. nov. differs from P.phoxocephalus by having large tubercles and warts on flanks and by its advertisement call. Calls of P.verrucolatus sp. nov. have one note (3–9 in P.phoxocephalus) that lasts more than the notes of the calls of P.phoxocephalus, and the dominant frequency, second harmonic frequency, and final and initial frequency of the notes are lower (Table 6; Fig. 6).

Variation.

Variation in live and preserved individuals is shown in Figures 26, 27. This section is based on 23 individuals of the QCAZ collection collected at the type locality and its surroundings. Photographs are available from 12 individuals. Coloration in life is mentioned in parenthesis. Skin on dorsum is shagreen with scattered prominent tubercles; dorsum bears a faint middorsal fold; a middorsal row of two or more tubercles is present on the head; lateral folds are inconspicuous or absent. Folds and tubercles in juveniles are more prominent and larger than those in adults. In preservative, middorsal fold, lateral folds, and tubercles on dorsum can be lost. Dorsal coloration varies from cream, light gray and light brown to dark brown (cream, pale yellow or light to dark brown with or without reddish or olive tones). Dorsum can have a scapular W-shaped marking, chevrons, longitudinal middorsal band, pale blotches, and black or white flecking. Dorsal markings in juveniles are more evident than those in adults. Groins, anterior and posterior surfaces of thighs, and concealed surfaces of tarsus and shanks are uniformly white (yellow) in juveniles. Adults bear dark brown to black reticulations outlining well-defined pale (yellow) spots. Dorsal surfaces of thighs bear dark oblique bars, well defined on juveniles, ill-defined or absent on adults. Ventral surfaces of thighs are white or cream (yellow). Venter is white with a light brown midline (reddish cream) extending to the throat; throat is white (cream, sometimes yellow in males). Iris is copper with black reticulations in adults; sclera varies from cream to light blue. Juveniles have a faint medial horizontal red streak.

Color variation in preserved individuals of Pristimantisphoxocephalus. A Dorsal view of (from left to right): QCAZ 58463 (female), QCAZ 58472 (female), QCAZ 58470 (male), QCAZ 58468 (male), QCAZ 58461 (male). B Ventral view of the same specimens. See Suppl. material 2 for locality data. All specimens are shown at the same scale.

https://binary.pensoft.net/fig/322419Advertisement call.

Based on recordings of two non-collected individuals at the type locality of P.phoxocephalus (November 17, 2014; 19h26; <10 °C). Advertisement calls of P.phoxocephalus consist of a series of 3–9 sharp notes (Fig. 6B). Notes last on average 0.154 s (range 0.133–0.175 s), silences between them, 0.135 s (range 0.110–0.159 s). The exact middle of the note presents the highest energy. The dominant and fundamental frequency of the notes is 2578 Hz. The note increases its frequency from beginning to end (2587–2452 Hz on average). Descriptive statistics for bioacoustic parameters are shown in Table 6.

Distribution, natural history, and conservation status.

Pristimantisphoxocephalus is known from its type locality, Pilaló, Cotopaxi Province, Ecuador, at 2340–2820 m a.s.l. (Fig. 2), which corresponds to Western Montane Forest. Individuals collected for this study were found by day inside bromeliads at 2–4 m above the ground. These bromeliads were on scattered trees within pastures. Lynch (1979) provides more ecological data for specimens collected at the type locality.

Currently, P.phoxocephalus is considered to be a Least Concern species (Rodríguez et al. 2004) under the assumptions: (i) Extent of Occurrence < 20000 km2, (ii) common and adaptable species with a presumed large population, (iii) unlikely to be declining fast enough to qualify for a more threatened category. After assessing the identity of this species, herein we assign it to the Critically Endangered Red List category following the B1ab(iii)+2ab(iii) IUCN criteria because: (i) it is known from one locality whose habitat is constantly degrading due to human settlements and cattle raising, (ii) Extent of Occurrence < 100 km2, (iii) Area of Occupancy < 10 km2.

Remarks.

Until now, P.phoxocephalus has been considered a single highly polymorphic species (e.g., Lynch 1979; Lynch and Duellman 1997; Duellman and Lehr 2009). We show that most populations previously ascribed to P.phoxocephalus represent other species (e.g., Pristimantisatillo, P.jimenezi, P.teslai sp. nov., P.torresi sp. nov., P.totoroi sp. nov., P.verrucolatus sp. nov., Pristimantis sp. (CCS2), Pristimantis sp. (UCS2), Pristimantis sp. (UCS3)). To determine the true identity of P.phoxocephalus, we made collections at the type locality (Pilaló, Cotopaxi Province, see Suppl. material 2 for the list of specimens). Surprisingly, sequences from Pilaló fell in two distinct clades, indicating the presence of two species. One juvenile (QCAZ 58425), collected at 2258 m, is actually P.totoroi sp. nov. All other specimens (e.g., QCAZ 58463) are P.phoxocephalussensu stricto. Examination of photographs of the holotype KU 142075 (Fig. 23A) showed unequivocally that it is conspecific with the specimens from the highland population. We included in our analysis sequences of “P.phoxocephalus” from two localities of Peru (Kañaris in Ferrenafe Province, Warmicocha-Cochapamba road in Chachapoyas Province); they do not belong to Huicundomantis (Suppl. material 3) and likely represent an undescribed species. Hence, P.phoxocephalus is endemic to Ecuador and has a restricted distribution.

Holotypes of Pristimantisteslai sp. nov. and P.torresi sp. nov. Photographs of preserved holotypes of AP.teslai (QCAZ 46213, male) and BP.torresi (QCAZ 47342, female). Dorsal view on the left, ventral view on the right. Specimens are shown at the same scale.

Pristimantisteslai is most similar to P.atillo, P.jimenezi, P.percultus, P.phoxocephalus, P.torresi sp. nov., P.totoroi sp. nov., and P.verrucolatus sp. nov. They share an acuminate and protruding snout with a keel at the tip. However, (except for P.percultus) P.teslai is unique among them by having the dorsum covered by prominent rounded tubercles. Additionally, lack of lateral folds distinguishes P.teslai from P.jimenezi, P.totoroi sp. nov., P.torresi sp. nov., and P.verrucolatus sp. nov. Groins of P.teslai are dark brown with yellow blotches irregularly bordered, different from those of P.atillo (groins orange surrounded or not by yellow blotches), P.jimenezi (pinkish, purplish or dark brown with small light brown to yellow spots), P.phoxocephalus (yellow with dark brown to black reticulations), and P.verrucolatus sp. nov. (reddish brown with light brown to yellow spots). In males, the tympanum diameter is significantly larger than that of P.totoroi sp. nov. (males Z = -2.63144, p = 0.0085, TD/SVL = 5.0–5.5% in P.teslai, 4.4–5.1% in P.totoroi sp. nov). Pristimantisteslai is smaller than P.verrucolatus sp. nov. (males Z = 2.35, p = 0.0188, SVL = 23.4–27.3 mm in P.teslai, 25.1–34.5 mm in P.verrucolatus sp. nov). It can be distinguished from P.percultus by the absence of cranial crests (low in P.percultus), the coloration of the iris (copper with thin reticulations in P.teslai; golden with wide black reticulations in P.percultus), and lacking the red labial stripe, characteristic of P.percultus.

Variation in preservative is shown in Figure 30, where all the type series is included. Coloration in life is known for two individuals; their photographs are shown in Figure 29. Coloration in life is in parenthesis. Background coloration of dorsal surfaces vary from gray to brown (olive to reddish brown); dorsum might bear dark brown W-shaped scapular marking, irregular chevrons, or longitudinal stripes (dorsum with red or cream irregular spots or blotches); head bears or not dark brown supratympanic stripe, canthal stripe, interorbital band and labial bars; flanks with or without black or white spots. Groins and anterior surfaces of thighs dark brown with pale (yellow) blotches irregularly bordered; posterior surfaces of thighs brown with pale (yellow) flecks; dorsal surfaces of thighs with irregular oblique pale (yellow) stripes, venter white to dusty white with scattered brown flecks (dusty white with or without faint yellow blotches). Iris is copper with thin black reticulations; light-blue sclera. Tubercles of the individual with the striped color pattern on dorsum are less prominent than those of the other individuals.

Pristimantisteslai is known from two Paramo localities in the eastern Andean slopes in Tungurahua Province (Fig. 2). Individuals were found beneath rocks, among moss or bunch grasses at day, or active on low vegetation up to 80 cm above ground at night. Parque Nacional Llanganates is, to a large extent, unexplored. It could have additional populations for this species. Given the scant available information, we assign P.teslai to the Data Deficient Red List Category (IUCN 2017).

Etymology.

The specific epithet is a noun in the genitive case and is a patronym for Nikola Tesla, a revolutionary inventor of the late 19th and early 20th century. It is named after him in recognition of his contributions to physics and his dedication to the ideal of providing free wireless electric power.

Remarks.

Pristimantisteslai has been mistakenly identified as P.phoxocephalus (e.g., collections at the QCAZ museum). Here, we recognize it as a different species and assign it to the P.phoxocephalus species group. Pristimantisteslai is most similar species to UCS1. They are sister species and have a genetic distance of 2.5%. Pristimantisteslai differs from UCS1 by having more prominent tubercles and smaller and fuzzier yellow blotches on the groins and posterior surfaces of thighs. However, these small differences may represent intraspecific variation. Hence, the status of UCS1 will remain tentative until additional specimens and populations of both species are examined.

Pristimantistorresi is similar to P.atillo, P.jimenezi, P.phoxocephalus, P.teslai, P.totoroi sp. nov., and P.verrucolatus sp. nov., which also have an acuminate snout with a fleshy keel. Pristimantisatillo usually has orange groins and black dots on the flanks, instead, P.torresi has brown groins with or without yellow spots, and its flanks lack black dots. The most similar species to P.torresi is P.jimenezi, whose iris varies from copper to red (golden to beige with a red medial streak in P.torresi). Pristimantistorresi differs from P.phoxocephalus in having a golden to beige iris (copper in P.phoxocephalus), brown groins with or without light brown to yellow spots (yellow with black reticulations in P.phoxocephalus), and a wider head relative to its body (males Z = -2.56285, p = 0.0104, HW/SVL = 33.4–34.4% in P.phoxocephalus, 34.2–37.8% in P.torresi; females Z = -2.08893, p = 0.0367, HW/SVL = 33.1–37.3% in P.phoxocephalus, 36.7–38.5% in P.torresi). Pristimantistorresi is easy to distinguish from P.teslai by having shagreen dorsal skin, lateral folds, and golden to beige iris with a red to reddish-brown medial streak (tuberculate dorsal skin, lateral folds absent and copper iris in P.teslai). Pristimantistotoroi sp. nov. has more prominent tubercles and folds than those of P.torresi, and its head is longer (males Z = 3.84623, p = 0.0001, HL/HW = 95.1–102.3% in P.torresi, 99.7–104% in P.totoroi sp. nov; females Z = -2.76079, p = 0.0058, HL/HW = 90.6–94.7% in P.torresi, 95.6–103.2% in P.totoroi sp. nov), relative to head width. Pristimantistorresi differs from Pristimantisverrucolatus sp. nov. in lacking large tubercles and warts on the flanks (present in P.verrucolatus) and having a golden to beige iris (coppery brown in P.verrucolatus).

Based on the 47 preserved specimens of the type series and photographs for 24 individuals. Variation in living and preserved individuals is shown in Figures 31, 32. Coloration in life is given in parenthesis. Dorsum coloration varies between cream, light gray and brown (cream, brown or orangey-brown). Some individuals present dark brown reticulations, chevrons, flecks, a middorsal band, an hourglass-shaped band or a thin white middorsal stripe on dorsum. Head always bears brown supratympanic stripes and interorbital stripes or bands; sometimes, it bears brown canthal stripes or labial bars. Limbs present faint or well-defined transversal bands. Flanks may have diagonal bars. Coloration of groins and concealed surfaces of hindlimbs has sexual dimorphism: both sexes have cream, gray or brown backgrounds (light purplish brown to medium brown); females have none or little pale (cream) flecks; males have small or big pale (yellow, cream or orangey yellow) spots, usually more conspicuous in larger males. Venter varies from white to dusty cream, usually with gray or light brown markings and a midline (in males, throat cream, light or bright yellow). Iris is golden to beige with black reticulations and a red or reddish-brown medial streak; sclera varies from cream to light blue. Coloration in life of each individual may change; the ground color of the hidden surfaces of thighs and other markings as the supratympanic stripe can vary from light purplish brown to medium brown in the same individual.

This species is known from the surroundings of Celica, Guachanamá and La Tolera, towns in the western Andean slopes of Loja Province in Ecuador (Fig. 2). It inhabits Inter-Andean Shrub region between 2101 and 2833 m a.s.l. Most individuals were found at night inside terrestrial or arboreal bromeliads up to 3 m above the ground, inside patches of native vegetation, or on low vegetation up to 60 cm above ground. During the day they were only found inside bromeliads. Calling males have been found on low vegetation, 30 cm above the ground in February.

We propose assigning P.torresi to the Critically Endangered Red List category following the B1ab(iii) IUCN criteria. Available records come from two localities (sensu IUCN 2017) whose habitat is being degraded by human settlements, cattle raising and agriculture; the Extent of Occurrence of the species < 100 km2 (21 km2).

Etymology.

The specific epithet is a noun in the genitive case and is a patronym for Omar Torres-Carvajal, curator of reptiles of Museo de Zoología at Pontificia Universidad Católica del Ecuador. The species name is in recognition of his significant contributions to herpetological research in Ecuador and the development of collections at the QCAZ museum.

Holotypes of Pristimantistotoroi sp. nov. and P.verrucolatus sp. nov. Photographs of preserved holotypes of AP.totoroi (QCAZ 25105, male) and BP.verrucolatus (QCAZ 46982, male). Dorsal view on the left, ventral view on the right. Specimens are shown at the same scale.

Pristimantistotoroi is similar to P.atillo, P.jimenezi, P.phoxocephalus, P.teslai, P.torresi and P.verrucolatus sp. nov., which also have an acuminate snout with a fleshy keel. The texture of its skin is different from the other species. Pristimantistotoroi has a shagreen dorsal skin, which is tuberculate in P.atillo; it lacks the large tubercles and warts on flanks present in P.verrucolatus sp. nov.; its tubercles and lateral folds are more prominent than those of P.atillo, P.jimenezi, P.phoxocephalus, and P.torresi. The coloration of the iris, golden with a red medial streak, helps distinguish P.totoroi from P.atillo (copper), P.jimenezi (copper to red), P.phoxocephalus (copper), P.teslai (copper), and P.verrucolatus sp. nov. (coppery brown). Additionally, the advertisement call of P.totoroi is different from the available calls of other species of the group. Notes of call of P.totoroi are shorter than those of P.jimenezi and P.verrucolatus sp. nov., inter-note intervals are longer than those of P.phoxocephalus, dominant frequency and frequency of the second harmonic are lower than those of P.jimenezi and higher than P.verrucolatus sp. nov. (Table 6).

Based on the 43 specimens of the type series and photographs of five individuals. Variation of live and preserved individuals is shown in Figures 34, 35. Coloration in life is given in parenthesis. Dorsal coloration varies from light to dark brown (tan to dark brown); middorsal fold is dark brown to black. Dorsum may bear irregular chevrons, a broad middorsal band or parallel longitudinal stripes; scattered black flecks or white spots may be present on dorsum; head bears a black or brown supratympanic stripe, sometimes an interorbital band or stripe, and labial bars; flanks usually bear dark diagonal bars. Dorsal surfaces of thighs bear pale transversal bands, sometimes formed by rows of pale spots. Groins and hidden surfaces of thighs are cream to brown (reddish cream to brown) with or without small pale flecks or spots. Venter is cream to dusty cream with a brown midline extending from the belly to the throat; brown or gray flecks on belly, chest, and throat may be present (in males, throat varies from white to yellow). Iris is golden with a medial horizontal red streak and fine black reticulations; sclera varies from white to cream. Skin ornamentation, as middorsal fold, dorsolateral folds, and dorsal tubercles, may fade in preservative.

Based on recordings of QCAZ 25105 (December 14, 2001; 20h00), and a non-collected individual from the type locality (December 14, 2001; 21h40; 10.2 °C). The advertisement call consists of a series of 2–6 sharp peep-like notes (Fig. 6C); each of these notes lasts about 0.13 s (range 0.10–0.15 s) and is separated of the following note by 0.34 s (range 0.31–0.37 s). The peak time occurs exactly in the middle of the note. Fundamental frequency is the same as dominant frequency, on average 2895 Hz (range 2776–3013 Hz). Frequency increases along the note (2551–2620 Hz on average). Descriptive statistics for bioacoustic parameters are shown in Table 6.

Distribution, natural history, and conservation status.

Pristimantistotoroi is known from Western Montane Forest of Bolívar, Chimborazo and Cotopaxi Provinces, between 2258–3200 m (Fig. 2). This species occurs in primary and secondary forests and pastures. Individuals found at night were on low vegetation up to 1.6 m above the ground or inside bromeliads up to 3 m above the ground. During the day they were found only inside bromeliads. Calling males have been found in October, December, and January on low vegetation during the night.

Following the B1ab(iii) + 2ab(iii) IUCN criteria, we consider P.totoroi to be Endangered because: (i) it is only known from three localities (sensu IUCN 2017); (ii) its habitat is gradually degraded by human settlements, cattle raising, and agriculture; (iii) its Extent of Occurrence < 5000 km2 (346 km2); and (iv) its Area of Occupancy <500 km2 (28 km2). Its distribution overlaps with one small protected area, Cashca Totoras Protected Forest. Unfortunately, the protection of the forest is not enforced and, as a result, it is being destroyed by logging.

Etymology.

The specific epithet is a noun in the genitive case that refers to the type locality of this species, the Cashca Totoras Protected Forest. This small reserve contains Western Montane Forest and Paramo natural regions. It is one of few protected areas in the western slopes of the Andes of central Ecuador, which are part of a biodiversity hotspot. Therefore, its effective protection is urgent to preserve unique assemblages of Andean biodiversity.

Remarks.

Pristimantistotoroi has been mistakenly referred to as P.phoxocephalus (e.g., Hedges et al. 2008). Here, we recognize it as a different species and assign it to the P.phoxocephalus species group.

Pristimantisverrucolatus is most similar to P.atillo, P.jimenezi, P.phoxocephalus, P.teslai, P.torresi, and P.totoroi. It differs from all of them by having thick lateral folds. Except for P.teslai, it is the only having large tubercles or warts on flanks; though tubercles on flanks are also present in P.teslai, they are not as large as the ones of P.verrucolatus; furthermore, dorsum of P.teslai is tuberculate, while the dorsum P.verrucolatus is shagreen, with or without scattered tubercles. Pristimantisverrucolatus is further distinguished from P.atillo by the coloration of its groins (orange in P.atillo; reddish brown with light brown to yellow spots in P.verrucolatus), and its larger size (males Z = 2.35, p = 0.0188, SVL = 23.4–27.3 mm in P.teslai, 25.1–34.5 mm in P.verrucolatus). Pristimantisjimenezi, which has an adjacent distribution at lower elevations, is smaller than P.verrucolatus (males Z = 3.58643, p = 0.0003, SVL = 21.8–27.0 mm in P.jimenezi, 25.1–34.5 mm in P.verrucolatus; females Z = 2.00347, p = 0.0451, SVL = 31.1–37.4 mm in P.jimenezi, 40.4–46.8 mm in P.verrucolatus). Additionally, the advertisement call of P.verrucolatus is very distinctive, distinguishing it from all species with available calls: P.jimenezi, P.phoxocephalus, and P.totoroi. The call of P.verrucolatus has a single note, longer than those of the calls of the other species. It also has the lowest dominant frequency and the highest variation in frequency from the beginning to the end of the note (Table 6). Pristimantis sp. (CCS1) is the closest species to P.verrucolatus (average p genetic distance = 3.2%), they are not morphologically alike. CCS1 has a chubbier body, lacks a keel at the tip of the snout (present in P.verrucolatus), has slightly expanded discs on fingers and toes (broadly expanded in P.verrucolatus), and lacks lateral folds (present in P.verrucolatus).

Data are based on 18 preserved specimens and photographs from six living individuals. Variation in life and preservative is shown in Figures 36, 37. Coloration in life is given in parenthesis. Dorsal coloration varies from light gray to dark gray or brown (light to dark brown). Dorsal pattern can be uniformly colored, with irregular reticulations or with a middorsal band. Light colored individuals bear a pale stripe on lips. Head usually bears a dark canthal stripe that continues along the upper eyelid and above the tympanum, and an interorbital stripe. Most specimens have labial bars. Flanks bear or not pale reticulations or black flecks. Groins and posterior surfaces of thighs are brown with cream dots (reddish brown with small light brown or orangey brown spots). Ventral surfaces of limbs are dusty cream to brown; venter is cream or white with or without brown reticulations; throat is cream to dusty brown with or without dark mottling. Iris is coppery brown with thin black reticulations; white sclera. Flanks have medium sized to large tubercles and warts, arranged or not in rows; lateral folds can be thick and continuous or be present as a row of tubercles.

Based on recordings of QCAZ 46981 (September 21, 2010; 19h00; 8.8 °C) and QCAZ 46984 (January 20, 2010; 21h10). Advertisement calls consist of a single sharp whistle (Fig. 6D); each lasting on average 0.43 s (range 0.41–0.46 s). The peak time of each note occurs exactly in the middle of its duration. The dominant and fundamental frequencies are the same, on average 2144 Hz (range 1927–2302 Hz). The note increases its frequency from beginning to end (2231–1838 Hz on average). Descriptive statistics for bioacoustic parameters are shown in Table 6.

Distribution, natural history, and conservation status.

This species is known from Western Andean slopes of Azuay Province, between 2943–3662 m a.s.l (Fig. 2). It inhabits Paramo and Western Montane Forest regions. Individuals collected during the day were found beneath rocks or inside arboreal bromeliads up to 2 m above ground; at night they were found active on branches of low shrubs, up to 80 cm above the ground, or inside bromeliads. Gravid females were found in August and January; calling males in January.

Pristimantisverrucolatus is known from only two localities (sensu IUCN) and has a very restricted distribution. These places are adjacent to Parque Nacional Cajas, a protected area with unexplored regions that represent potential distribution for the species. Thus, following the IUCN (2017) guidelines, we assign this species to the Data Deficient Red List category.

Etymology.

The specific epithet is a noun in apposition with masculine gender. It is derived from the Latin words verruca meaning wart, and latus meaning flanks. The name refers to the large and low tubercles or warts on flanks that characterize this species.

Content. The P.cryptomelas species group comprises four described species, P.cryptomelas, P.gagliardoi, P.muscosus, and P.spinosus, and the newly described P.nangaritza sp. nov. We suspect that the species content of the group will increase with collections and genetic studies of populations from northern Peru.

Distribution. Members of the P.cryptomelas group occur in the eastern Andean slopes of southern Ecuador and northern Peru. In Ecuador, they inhabit the Eastern Foothill and Montane Forest of Cañar, Loja, Morona Santiago, and Zamora Chinchipe Provinces, between elevations of 1800 and 3500 m a.s.l. In Peru, they live between 1770 and 2820 m a.s.l. Distribution based on QCAZ collection specimens, Duellman and Lehr (2009), and Yánez-Muñoz et al. (2012).

Remarks. The P.muscosus specimen included in our phylogeny is from the same and only population of P.muscosus reported for Ecuador (Yánez et al. 2012). The population is 216 km from the type locality in Peru (east slope of Abra Pardo de Miguel). Our results indicate that most species of Huicundomantis have small geographic ranges. The large geographic distance from the type locality of P.muscosus suggests that the Ecuadorian population may represent an undescribed species. The same applies to Peruvian populations of P.cryptomelas, which are widely separated from the type locality in Ecuador.

It is most similar to P.cryptomelas, P.gagliardoi, P.muscosus, P.spinosus, and P.versicolor. The postocular folds of P.nangaritza are lower than those of P.cryptomelas, the background color of the posterior surfaces of thighs is light brown instead of black, its body is smaller (Table 5), and males have vocal slits (absent in males of P.cryptomelas). Pristimantisgagliardoi can be distinguished from P.nangaritza by having W-shaped postocular folds (“) (”-shaped in P.nangaritza), smaller tympanum (Table 5), and by lacking vocal slits. Pristimantismuscosus has smaller tubercles on the upper eyelid (round in P.muscosus; subconical in P.nangaritza), lacks tubercles on the inner edge of tarsus (present in P.nangaritza), and have dark brown or black groins. Pristimantisspinosus differs from P.nangaritza in having low cranial crests (absent in P.nangaritza) and black posterior surfaces of thighs with white spots. Pristimantisversicolor is readily recognized from P.nangaritza by lacking postocular folds and having a proportionally larger tympanum (females Z = 2.16506, p = 0.0304 TD/SVL = 4.7–5.9% in P.nangaritza, 5.2–7.2% in P.versicolor; males Z = 3.45394, p = 0.0006, TD/SVL = 5.1–5.4% in P.nangaritza, 5.8–6.4% in P.versicolor).

This section is based on 18 specimens of the type series. In preservative, dorsum varies from light to dark brown with darker markings including a scapular W, irregular chevrons, and interorbital stripe. Some individuals have a pattern of longitudinal parallel stripes. Posterior surfaces of thighs are brown with or without minute pale flecks. Groins with the same coloration as posterior surfaces of thighs or venter. Venter cream with varying amount of brown mottling. This variation is shown in Figure 39. Coloration in life is unknown.

Distribution, natural history, and conservation status.

Pristimantisnangaritza is only known from its type locality, Alto Nangaritza Protected Forest, Zamora Chinchipe Province, Ecuador, a low vegetation forest with bromeliads, orchids, moss, and Podocarpus trees that belongs to the Eastern Foothill Forest, between 1809 and 1843 m a.s.l. (Fig. 1). Individuals were found active at night on branches, 1–2 m above ground. Calling males were found in April.

We consider it as a Data Deficient species because adjacent areas in Alto Nangaritza Protected Forest are difficult to access and poorly explored.

Etymology.

The specific epithet refers to the type locality of this species, Alto Nangaritza Protected Forest. This protected area preserves native vegetation of the Cordillera del Cóndor and hosts unique geologic formations in Ecuador, called Tepuyes. This reserve is largely unexplored and is currently threatened by the potential opening of roads for mining activities; 80% of its territory is under mining concessions.

On the identity of Pristimantisriveti

Several species described here were previously misidentified as P.riveti, including P.lutzae, P.gloria, P.multicolor, P.chomskyi, and Pristimantis sp. (CCS1) (e.g., Almendáriz and Orcés 2006; Heinicke et al. 2007; Padial et al. 2014). These misidentifications were based on a redescription of “P.riveti” by Lynch (1979). Populations used for Lynch’s redescription (Azuay Province, Cuenca) are geographically distant (400 km to the south) from the type locality of P.riveti, “Mirador”, which, based on the collector itinerary, we infer to be in Carchi Province, Ecuador. Although Despax (1911) did not mention the province for the type locality, the holotype was collected by Paul Rivet during the Second Geodesic Mission which visited “Mirador” in Carchi Province (Perrier 1913). According to Frost (2019), Mirador is in Tungurahua Province, Ecuador, but we could not find a reference to support that statement.

The great distance between the type locality of P.riveti and populations in Azuay Province used in Lynch’s redescription indicate that they are not conspecific. This conclusion is consistent with morphological comparisons of photographs of the holotype of P.riveti, MNHNP 1902.357 (Fig. 23B) with P.lutzae (= “P.riveti” of Lynch 1979) which showed marked differences. The holotype of P.riveti is an adult female whose snout-vent length is 34.2 mm, a size above the range for females of Pristimantislutzae (29.7–33.9 mm); Pristimantisriveti has distinctive big white irregular blotches dispersed over the dorsal surface, which are absent in P.lutzae; besides, Toe V is slightly longer than toe III in P.riveti while it is longer or much longer than Toe III in P.lutzae. The combined evidence indicates that the populations from Azuay usually ascribed to P.riveti represent a previously undescribed species that we have named here.

Based on information published by Perrier (1913) we were able to infer the position of the type locality of P.riveti. Mirador is a mountain peak at an elevation of 3830 m near the border between Carchi and Sucumbíos Provinces. Recent collections made on that mountain, at an elevation between 3500 and 3792 m, include specimens similar to the holotype of P.riveti (QCAZ 69853, 69855, 69857–859, 69861, 69865–869). We tentatively considered these specimens to be the only documented records of P.riveti sensu stricto besides that of the holotype.

DiscussionCryptic diversity within Huicundomantis

Herein, we document the existence of 16 new candidate species within a clade of which only 12 species were previously described. These results represent an increase in species richness of 133%. Moreover, several populations not included in our study likely represent additional undescribed species. For example, in the QCAZ collection, specimens from 6 km E Loja, Loja Province (e.g., QCAZ 21708) or Zamorahuaico, Zamora Chinchipe Province (e.g., QCAZ 22529) are morphologically similar to P.phoxocephalus and appear to represent undescribed species. These populations were excluded from our review because they lacked tissues for DNA analyses. Similarly, several populations of “P.phoxocephalus” from southern Ecuador reported by Lynch (1979) (e.g., 10 km SW Victoria del Portete, Azuay Province; 18.4 km NW El Tambo, Cañar Province; Saraguro, Loja Province) and from northern Peru by Duellman and Lehr (2009), Duellman and Pramuk (1999), and Duellman and Wild (1993) are likely undescribed species as indicated by our samples of “P.phoxocephalus” from Peru which fall outside Huicundomantis. Given the branch lengths that separate them, the three Peruvian samples appear two represent two different species (Suppl. material 3). Our results suggest that Andean Pristimantis still hide a large number of cryptic undescribed species. If the content of undescribed species of our group is a representative sample of all Pristimantis, we would expect the discovery of hundreds of new species in the coming decades.

Characters that help diagnose species of Huicundomantis are body tuberculation, the presence of dermal folds (e.g., middorsal, lateral, postocular folds), and the coloration in life of the groins, concealed surfaces of thighs, and iris. These characters are frequently lost in preservative, which could explain why previous taxonomic reviews, mainly based on morphology of preserved specimens, lumped so many species into single binomens. Hence, we highlight the importance of documenting coloration and skin texture in live individuals, prior to preservation. We also found extensive intraspecific and even intrapopulation morphological variation, especially in coloration and skin texture (e.g., P.multicolor; Fig. 24). This variation can only be properly described by sampling a large number of individuals. Future taxonomic reviews of Pristimantis will greatly benefit from documenting both life coloration and genetic variation in a large number of specimens.

Morphometrics were of little help to diagnose species except for several species pairs. For morphometric diagnoses, we compared body size and head proportions, which can be useful taxonomic characters (Arroyo et al. 2005). The addition of other measurements or the use of new approaches such as geometric morphometrics (Kaliontzopoulou 2011; Acevedo et al. 2016) could help to better differentiate cryptic species of Huicundomantis.

Similar to morphometrics, environmental envelopes were of limited help to distinguish closely related species. However, we were able to discriminate among species inhabiting high-altitude Paramo habitats from those inhabiting warmer regions in Eastern Foothill Forest, Deciduous Costa Forest, and Western Foothill Forest. These environmental differences are mirrored by distinct body types that appear to be related to different environments. Species from paramo habitats have a chubby appearance and usually have narrow finger discs while species from lower altitudes have a slenderer body and wider discs.

When available, advertisement calls allowed unequivocally diagnosing of closely related species. This is congruent to previous studies in Pristimantis (Hutter and Guayasamin 2015), and other Neotropical frogs (e.g., Padial et al. 2008; Funk et al. 2012; Caminer et al. 2014,) in which divergence in advertisement calls led to the discovery of cryptic species. Advertisement calls are under sexual selection and were the most variable trait between species. Therefore, it seems likely that sexual selection played an important role in speciation of Huicundomantis.

Huicundomantis, a new subgenus for Pristimantis

We propose Huicundomantis as a subgenus given that it has strong phylogenetic support and morphological distinctiveness. Currently, two subgenera are recognized within the genus Pristimantis: Pristimantis and Hypodictyon. According to recent reviews, Pristimantis is paraphyletic while Hypodictyon (sensu Crawford et al. 2010) is monophyletic (Heinicke et al. 2017). We agree with Heinicke et al. (2017) on the need for the recognition of new subgenera within Pristimantis to convey phylogenetic information among its 532 species. Padial et al. (2014) did not recognize the subgenus Pristimantis given its rampant paraphyly. However, the recognition of Hypodictyon is justified given its well established monophyly. The description of the new monophyletic subgenus Huicundomantis is an additional step to reach the goal of splitting Pristimantis into monophyletic subgenera. Moreover, Huicundomantis does not generate taxonomic instability because the subgenus Pristimantis has been largely ignored as a result of its nearly complete redundancy with its parent genus. Finally, we highlight that, if needed, the species content of Huicundomantis can be redefined to accommodate the requirements of a large-scale taxonomic review of its parent genus.

The name Huicundomantis refers to the association of most of its species to bromeliad plants (huicundos in Quechua). This association has been reported, at least facultatively, in P.atillo, P.atratus, P.chomsky, P.cryptomelas, P.jimenezi, P.multicolor, P.phoxocephalus, P.tinguichaca, P.torresi, P.totoroi, P.verrucolatus, and P.versicolor (Ron et al. 2019 and references therein). Another clade of Pristimantis with a large number of species associated to bromeliads is the P.lacrimosus group (Hedges et al. 2008). Both clades are not closely related (Pyron 2014), which suggests that the bromeliad association evolved independently in both groups. Numerous other species of Pristimantis of uncertain phylogenetic position also occur in bromeliads (e.g., P.huicundo, P.orphnolaimus). Understanding the evolution origin of this ecological character will require a comprenhensive analysis across all species of Pristimantis.

Speciation and genetic divergence

We found seven sister species pairs (including unconfirmed candidate species) within Huicundomantis. In all of them, their distribution ranges do not overlap and do not have significant differences in environmental envelope. Allopatric distribution and lack of differences in environmental envelope suggest that speciation has been mainly driven by incidental divergence in allopatry, rather than ecological divergence along environmental gradients (Peterson et al. 1999; Graham et al. 2004). The fragmented landscape of the Andean slopes, and expansions and contractions of the distribution range due to environmental oscillations should frequently result in geographic isolation, making allopatric speciation the most likely mode of speciation for Huicundomantis. Moreover, Andean slopes allow the existence of high diversity and endemism because they present sharp gradients of elevation, temperature, and precipitation. These heterogeneous conditions should explain the small distribution ranges observed in montane species of frogs (Wollenberg et al. 2008). The pattern of species richness and diversification of Huicundomantis is congruent with other studies from mountainous areas, which report allopatric speciation as the most frequent mode of speciation in the group (e.g., Lynch and Duellman 1997; Coloma et al. 2012; Jungfer et al. 2013).

We found that the 3% genetic distance threshold (gene 16S) proposed to identify candidate species (Fouquet et al. 2007; Vieites et al. 2009) could be too conservative. In Huicundomantis, the minimum genetic distance between confirmed candidate species was 2.0%; applying the 3% threshold would have resulted in 3 confirmed candidate species overlooked. Our results are similar to those of other systematic reviews of Neotropical amphibians (e.g., Padial and de la Riva 2009; Coloma et al. 2012; Funk et al. 2012; Jungfer et al. 2013; Caminer and Ron 2014; Ortega et al. 2015) showing that the 3% genetic distance threshold could result in underestimation of species numbers.

Cryptic diversity and conservation

The discovery of cryptic diversity can impact estimates of the conservation status of species (Bickford et al. 2007). One of the main variables to assess the conservation status of species is the size of its distribution range (IUCN 2017). After dividing what was thought to be a single species into several cryptic species, distribution ranges of the resulting species are smaller than those of the original; therefore, the conservation status should be revaluated after cryptic species are discovered. For example, until now, Pristimantisphoxocephalus was considered to be distributed along the western and eastern Andean slopes of Ecuador and Peru (elevations 1800–3100 m a.s.l; Rodríguez et al. 2004). Given its large distribution range, it was considered Least Concern by the IUCN Red List (Rodríguez et al. 2004). Our review shows that P.phoxocephalus is known from a single locality, Pilaló, in the western Andean slopes of central Ecuador. Therefore, we propose that P.phoxocephalus is Critically Endangered according to the IUCN (2017) guidelines.

https://binary.pensoft.net/file/322432This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Nadia B. Páez, Santiago R. Ron10.3897/zookeys.868.26766.suppl233641808E002865-CF8C-568C-8D26-9CCA25CE0347Supplementary material 2

https://binary.pensoft.net/file/322433This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Nadia B. Páez, Santiago R. Ron10.3897/zookeys.868.26766.suppl333641820AF0E7DD-808F-580B-8CC3-3D0859811903Supplementary material 3

Phylogram of Huicundomantis including outgroup

Data type: phylogram

Explanation note: ML tree for genes 16S, ND1 and RAG1. Bootstrap values (%) are shown under the corresponding branches; missing values indicate values below 50 %. The number of collection, identification, country and locality of the samples are shown next to each terminal. EC is for Ecuador, PE for Peru, NP is for National Park, PF is for Protected Forest. Terminals pointed with an arrow correspond to specimens from Peru previously ascribed to P.phoxocephalus. Figure includes only outgroup within Pristimantis. See vouchers and GenBank accession numbers of all sequences used for molecular analyses in Table 1.

https://binary.pensoft.net/file/322434This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Nadia B. Páez, Santiago R. Ron10.3897/zookeys.868.26766.suppl4336418436ED37D6-EAE0-5A06-B90A-4681042E60A2Supplementary material 4

Mitochondrial phylogram depicting relationships within Huicundomantis

Data type: phylogram

Explanation note: ML tree for genes 16S and ND1. Bootstrap values (%) are shown under the corresponding branches; missing values indicate values below 50 %; asterisks indicate support values of 100%. The number of collection, identification, country and locality of the samples are shown next to each terminal. EC is for Ecuador, PE for Peru, PA is for Panama, NP is for National Park, PF is for Protected Forest. Outgroup is shown.

https://binary.pensoft.net/file/322435This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Nadia B. Páez, Santiago R. Ron10.3897/zookeys.868.26766.suppl53364186C802DB59-89A1-50B2-BFCB-4E1D7399E02ASupplementary material 5

Phylogram depicting relationships within Huicundomantis

Data type: phylogram

Explanation note: ML tree for nuclear gene RAG1. Bootstrap values (%) are shown under the corresponding branches; missing values indicate values below 50 %. The number of collection, identification, country and locality of the samples are shown next to each terminal. EC is for Ecuador, PE for Peru, PA for Panama, NP is for National Park, PF is for Protected Forest. Outgroup is shown.

https://binary.pensoft.net/file/322436This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.Nadia B. Páez, Santiago R. Ron